Anti-hepcidin antibodies and uses thereof

ABSTRACT

The present application relates to antibodies that specifically bind to hepcidin and methods of using the antibodies. Another aspect relates to antibodies which bind hepcidin and regulate iron homeostasis. Another aspect relates to the use of humanized antibodies which bind hepcidin for the treatment of a disease or condition associated with hepcidin.

CROSS REFERENCE

This application is a divisional of U.S. patent application Ser. No.14/771,135, filed Aug. 27, 2015, now U.S. Pat. No. 9,657,098; which is aU.S. §371 National Stage Entry of International Application No.PCT/US2014/026804, filed Mar. 13, 2014; which claims the benefit ofpriority from U.S. Provisional Application No. 61/791,953, filed Mar.15, 2013, which application is incorporated herein by reference in itsentirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 7, 2014, isnamed 44546-702.601_SL.txt and is 38,025 bytes in size.

BACKGROUND OF THE INVENTION

Iron is an essential trace element required for growth and developmentof living organisms. In mammals, iron content is regulated bycontrolling iron absorption, iron recycling, and release of iron fromthe cells in which it is stored. Iron is predominantly absorbed in theduodenum and upper jejunum by enterocytes. Iron is recycled fromdegraded red cells by reticuloendothelial macrophages in bone marrow,hepatic Kupffer cells and spleen. Iron release is controlled byferroportin, a major iron export protein located on the cell surface ofenterocytes, macrophages and hepatocytes, the main cells capable ofreleasing iron into plasma. Hepcidin binds to ferroportin and decreasesits functional activity by causing it to be internalized from the cellsurface and degraded. (Nemeth et al., Science, 306:2090-3, 2004).

SUMMARY OF THE INVENTION

Provided herein are antibodies and antigen-binding fragments thereofthat bind to hepcidin (Hep) or a hepcidin peptide. In one aspect,provided herein is an antibody, or antigen-binding fragment thereof,that specifically binds to the N-terminus of hepcidin or a hepcidinpeptide and neutralizes the activity of hepcidin in vitro and/or invivo.

In one aspect, provided herein is an antibody, or antigen-bindingfragment thereof, that specifically binds to hepcidin or a hepcidinpeptide, comprising a heavy chain variable region and a light chainvariable region,

wherein said heavy chain variable region comprises:

(i) a CDR1 having an amino acid sequence of any one of SEQ ID NOS:55-57,

(ii) a CDR2 having an amino acid sequence of any one of SEQ ID NOS:58-60, and

(iii) a CDR3 having an amino acid sequence of any one of SEQ ID NOS:61-63;

and said light chain variable region comprises:

(i) a CDR1 having an amino acid sequence of any one of SEQ ID NOS:64-66,

(ii) a CDR2 having an amino acid sequence of any one of SEQ ID NOS:67-69, and

(iii) a CDR3 having an amino acid sequence of any one of SEQ ID NOS:70-72.

In one aspect, provided herein is an antibody, or antigen-bindingfragment thereof, that specifically binds to hepcidin or a hepcidinpeptide, comprising a heavy chain variable region and a light chainvariable region,

wherein said heavy chain variable region comprises:

(i) a CDR1 having an amino acid sequence encoded by any one of SEQ IDNOS: 1-3,

(ii) a CDR2 having an amino acid sequence encoded by any one of SEQ IDNOS: 4-6, and

(iii) a CDR3 having an amino acid sequence encoded by any one of SEQ IDNOS: 7-9;

and said light chain variable region comprises:

(i) a CDR1 having an amino acid sequence encoded by any one of SEQ IDNOS: 10-12,

(ii) a CDR2 having an amino acid sequence encoded by any one of SEQ IDNOS: 13-15, and

(iii) a CDR3 having an amino acid sequence encoded by any one of SEQ IDNOS: 16-18.

In one aspect, an antibody, or antigen-binding fragment thereof,provided herein comprises IgG1 or an IgG4 variable heavy chain andvariable light chain.

In one aspect, provided herein is an antibody, or antigen-bindingfragment thereof, that specifically binds to hepcidin or a hepcidinpeptide, that is prepared by injecting a rodent (i.e., mouse, rat orrabbit) with a peptide having an amino acid sequence of any one of SEQID NOS: 19-27. In another embodiment, the peptide is conjugated to acarrier (e.g., keyhole limpet hemocyanin (KLH)) or administered with anadjuvant (complete Freund's adjuvant (CFA) or incomplete Freund'sadjuvant (IFA)). In another embodiment the peptide is conjugated to ahapten (e.g., dinitrophenol [DNP]) and a carrier.

A hepcidin peptide to which an antibody, or antigen-binding fragmentthereof, binds may have, in some instances, an amino acid sequence ofSEQ ID NO: 19.

Provided herein is an antibody, or antigen-binding fragment thereof,that specifically binds to an epitope comprising amino acid sequence ofany one of Hep-5, Hep-9, Hep-20, Hep 22 and Hep25.

In one embodiment, the antibody, or antigen-binding fragment thereof,specifically binds to an epitope comprising an amino acid sequence ofHep-20 (SEQ ID NO: 22), Hep 22 (SEQ ID NO: 23) and Hep25 (SEQ ID NO:19).

In one embodiment, the antibody, or antigen-binding fragment thereof,specifically binds to an epitope comprising Hep-5 (SEQ ID NO: 25) orHep-9 (SEQ ID NO: 24). In another embodiment, provided herein is anantibody, or antigen-binding fragment thereof, that specifically bindsto an epitope comprising amino acid residues 1-9 of hepcidin. In anotherembodiment, the antibody, or antigen-binding fragment thereof,specifically binds to 2, 3, 4, 5, 6, 7, 8, or 9 amino acid residues ofan epitope comprising amino acid residues 1-9 of hepcidin.

In another embodiment, the antibody, or antigen-binding fragmentthereof, is monoclonal antibody comprising a heavy chain CDR1 encoded bySEQ ID NO: 55, a heavy CDR2 encoded by SEQ ID NO: 58, a heavy chain CDR3encoded by SEQ ID NO: 61, a light chain CDR1 encoded by SEQ ID NO: 64, alight CDR2 encoded by SEQ ID NO: 67, and a light chain CDR3 encoded bySEQ ID NO: 70.

In another embodiment, the antibody, or antigen-binding fragmentthereof, is monoclonal antibody comprising a heavy chain CDR1 encoded bySEQ ID NO: 56, a heavy CDR2 encoded by SEQ ID NO: 59, a heavy chain CDR3encoded by SEQ ID NO: 61, a light chain CDR1 encoded by SEQ ID NO: 65, alight CDR2 encoded by SEQ ID NO: 68, and a light chain CDR3 encoded bySEQ ID NO: 71.

In another embodiment, the antibody, or antigen-binding fragmentthereof, is monoclonal antibody comprising a heavy chain CDR1 encoded bySEQ ID NO: 57, a heavy CDR2 encoded by SEQ ID NO: 60, a heavy chain CDR3encoded by SEQ ID NO: 63, a light chain CDR1 encoded by SEQ ID NO: 66, alight CDR2 encoded by SEQ ID NO: 69, and a light chain CDR3 encoded bySEQ ID NO: 72.

The antibody may be, for example, a monoclonal antibody, a chimericantibody, a human antibody, or a humanized antibody. In one embodiment,a humanized variable heavy chain comprises an amino acid sequence setforth as SEQ ID NO: 40. In another embodiment, a humanized variablelight chain comprises an amino acid sequence set forth as SEQ ID NO: 38.

In one aspect, provided herein is an antibody, or antigen-bindingfragment thereof, comprises a heavy chain variable region frameworkregion; and a light chain variable region framework region as set forthin the Sequence Listing below where the CDRs identified in any one ofSEQ ID NOS: 1-18 are inserted into the framework region utilizing Kabatnumbering.

The antigen-binding fragment may be, for example, a Fab fragment, a Fab′fragment, a F(ab′)2 fragment, an Fv fragment, an scFv fragment, a singlechain binding polypeptide, a Fd fragment, a variable heavy chain, avariable light chain or a dAb fragment. An antigen-binding fragment maybe, for example, an AVIMER, a diabody, or a heavy chain dimer. A heavychain dimer may be, for example, a camelid or a shark heavy chainconstruct.

An antibody, or antigen-binding fragment thereof, described herein mayhave a dissociation constant (Kd) of about 1 to about 10 pM, from about10 to about 20 pM, from about 1 to about 29 pM, from about 30 to about40 pM, from about 10 to about 100 pM, or from about 20 to about 500 pM.

An antibody, or antigen-binding fragment thereof, described herein mayhave a dissociation constant (Kd) of less than about 500 pM, less thanabout 400 pM, less than about 300 pM, less than about 200 pM, less thanabout 100 pM, less than about 75 pM, less than about 50 pM, less thanabout 30 pM, less than about 25 pM, less than about 20 pM, less thanabout 18 pM, less than about 15 pM, less than about 10 pM, less thanabout 7.5 pM, less than about 5 pM, less than about 2.5 pM, or less thanabout 1 pM.

An antibody, or antigen-binding fragment thereof, described herein mayhave an affinity for hepcidin or a hepcidin peptide of from about 10⁻⁹to about 10⁻¹⁴, from about 10⁻¹⁰ to about 10⁻¹⁴, from about 10⁻¹¹ toabout 10⁻¹⁴, from about 10⁻¹² to about 10⁻¹⁴, from about 10⁻¹³ to about10⁻¹⁴, from about 10⁻¹⁰ to about 10⁻¹¹, from about 10⁻¹¹ to about 10⁻¹²,from about 10⁻¹² to about 10⁻¹³, or 10⁻¹³ to about 10⁻¹⁴.

Provided herein is a composition, comprising an antibody, orantigen-binding fragment, described herein, and an acceptable carrier orexcipient.

Also provided herein is an isolated nucleic acid molecule comprising anucleotide sequence that encodes an antibody, or antigen-bindingfragment thereof, described herein. Also provided herein is anexpression vector comprising the nucleic acid molecule, operably linkedto a regulatory control sequence. Also provided herein is a host cellcomprising a vector or a nucleic acid molecule provided herein. Alsoprovided herein is a method of using the host cell to produce anantibody, comprising culturing the host cell under suitable conditionssuch that the nucleic acid is expressed to produce the antibody.

Provided herein are therapeutic methods utilizing an antibody orantigen-binding fragment thereof, described herein. In one aspect,provided herein is a method of treating a disorder of iron homeostasisin a subject in need thereof, comprising administering to said subject acomposition described herein. In another aspect, provided herein is amethod of modulating hepcidin activity in a subject in need thereof,comprising administering to said subject a composition described herein.In yet another aspect, provided herein is a method for treating adisorder of iron homeostasis in a subject in need thereof, comprisingadministering to said subject a composition described herein. In yetanother aspect, provided herein is a method of treating hemochromatosisin a subject in need thereof, comprising administering to said subject acomposition described herein. In yet another aspect, provided herein isa method of treating a subject with pathologically or inappropriatelyelevated levels of hepcidin (inappropriately elevated relative to bodyand plasma iron stores), comprising administering to said subject apharmaceutical composition described herein. In yet another aspect,provided herein is a method of treating anemia in a subject in needthereof, comprising administering to said subject a compositiondescribed herein. In yet another aspect, provided herein is a method oftreating or reducing inflammation in a subject in need thereof,comprising administering to said subject a composition described herein.In one embodiment, inflammation to be treated or reduced is chronicinflammation. In yet another aspect, provided herein is a method oftreating an inflammatory disease in a subject in need thereof,comprising administering to said subject a composition described herein.In yet another aspect, provided herein is a method of treating aninfection in a subject in need thereof, comprising administering to saidsubject a composition described herein. An infection may be, forexample, a bacterial, fungal, or viral infection. In yet another aspect,provided herein is a method of treating Iron refractory iron deficiencyanemia (IRIDA). In yet another aspect, provided herein is a method oftreating Anemia of Inflammation (AI) and Anemia of Chronic Disease(ACD). In yet another aspect, provided herein is a method of treatingchronic kidney disease (CKD). In yet another aspect, provided herein isa method of treating cancer and Chemotherapy Induced Anemia (CCIA) whichare associated with elevated hepcidin. In yet another aspect, providedherein is a method of treating neuro-inflammatory diseases which areassociated with elevated hepcidin.

Any of such methods may, in some instances, further compriseadministering to said subject one or more erythropoiesis stimulators.Erythropoiesis stimulators include, but are not limited to,erythropoietin, an erythropoietin variant, an erythropoiesis stimulatingagent (ESA; such as, for example, Epoetin alfa [e.g., Procrit®, Epogen®,etc.], Epoetin beta [e.g., NeoRecormon, etc.], Darbepoetin alfa [e.g.,Aranesp®, etc.], Methoxy polyethylene glycol-epoetin beta [e.g.,Mircera®, etc.], etc.), a hypoxia inducible factor (HIF) prolylhydroxylase inhibitor, a bone marrow derived erythroid factor (e.g.erythroferrone), a mini-hepcidin peptide (see, e.g., U.S. PublicationNo. 20120040894, by Ganz et al., which is incorporated herein byreference), an antisense inhibitor of hepcidin (see, e.g., U.S.Publication No. 20100136015, by Lin and Babitt., which is incorporatedherein by reference), a siRNA inhibitor of hepcidin (Id.), miRNAinhibitor of hepcidin (Id.), an anti-BMP-2 antibody (Id.), an anti-BMP-4antibody (Id.), an anti-BMP-6 antibody (Id.), a small molecule inhibitor(Id.), an anti-IL-6 antibody (see, e.g., U.S. Publication No.20110059080, by Cornfeld et al., which is incorporated herein byreference), an anti-TNF-alpha antibody, methotrexate, ananti-inflammatory agent (e.g., a steroid [e.g., a corticosteroid, etc.];a non-steroidal inflammatory drug [NSAID; e.g., aspirin, ibuprofen,naproxen, a cyclooxygenase (COX) enzyme inhibitor, etc.], a hormone(e.g. testosterone), or an immune selective anti-inflammatory derivative[ImSAID; e.g., tripeptide FEG (Phe-Glu-Gly) and its D-isomer feG]),hemojuvelin, an antibody that binds erythropoietin, and combinationsthereof. In one embodiment, the antibody, or antigen-binding fragmentthereof, that specifically binds hepcidin and the erythropoiesisstimulator are administered concurrently or sequentially.

Administration of a composition herein may be by any suitable meansincluding, but not limited to, injection. In one embodiment, injectionmay be, for example, intravenous, subcutaneous, intramuscular injection,or spinal injection into the cerebrospinal fluid (CSF).

Provided herein is a container means comprising a composition describedherein. The container means may be any suitable container which mayhouse a liquid or lyophilized composition including, but not limited to,a vial, syringe, bottle, an in intravenous (IV) bag or ampoule. Asyringe may be able to hold any volume of liquid suitable for injectioninto a subject including, but not limited to, 0.5 cc, 1 cc, 2 cc, 5 cc,10 cc or more.

Provided herein are kits, comprising a composition or compositionsdescribed herein. In one aspect, provided herein is a kit for treating adisorder associated with elevated hepcidin levels or a disorder of ironhomeostasis, comprising an antibody, or an antigen-binding fragmentthereof, as described herein and an erythropoiesis stimulator. It wouldbe understood, in some instances, that hepcidin can be in the normalrange but inappropriately elevated relative to iron stores.

In another aspect, provided herein is a kit for treating a disorderassociated with elevated hepcidin levels or a disorder of ironhomeostasis, comprising an antibody, or an antigen-binding fragmentthereof, as described herein, and a label attached to or packaged withthe container, the label describing use of the antibody, or anantigen-binding fragment thereof, with an erythropoiesis stimulator.

In another aspect, provided herein is a kit for treating a disorderassociated with elevated hepcidin levels, comprising an erythropoiesisstimulator and a label attached to or packaged with the container, thelabel describing use of the erythropoiesis stimulator with an antibody,or an antigen-binding fragment thereof, as described herein.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1. Examples of hepcidin peptide antigen sequences used to immunizeBALB/c mice for hybridoma production and discovery of MAbs H32, 583, and1B1.

FIG. 2. Example of a first round screen of isolated hybridomas usingneutravidin coated plates coated with K18-biotin hepcidin-25 anddetected by anti-mouse IgG (H+L) conjugated HRP. Displayed are opticaldensities (OD) at 450 nm following HRP development and stop solutionaddition. Note positive signals (>2.0 OD) in Column 9 rows D-F. Positiveand negative control wells are G12 and H12, respectively.

FIG. 3. Example of a second round screen of isolated hybridomas fromfirst round screens determined to be positive (FIG. 2) using rabbitanti-mouse Fc coated plates to capture mouse IgG from hybridomasupernatants. Bound mouse IgGs are then screened for binding toK18-biotin hepcidin-25 using streptavidin conjugated HRP. Displayed areoptical densities (OD) at 450 nm following HRP development and stopsolution added. Note positive signals (>2.0 OD) in Column 9 row D.Positive and negative control wells are G12 and H12, respectively.

FIG. 4. Screening for functional activity of hybridomas positive foranti-hepcidin binding. Wells were coated with anti-mouse Fc antibodiesand blocked. In duplicate wells, each indicated hybridoma supernatantwas added to binding buffer containing 1 ng NT-biotin hepcidin-25tracer, with or without 100 ng synthetic hepcidin-25. Binding of theNT-biotin hepcidin-25 was detected with SA-HRP as OD at 450 nm afteraddition of stop solution to the wells. Antibody 5A3 (as did 4B1 and5A4) showed excellent binding in buffer without hepcidin-25 and wascompleted blocked by hepcidin-25 in the binding buffer indicatinghybridoma 5A3 contained an antibody that bound to both NT-biotinhepcidin-25 and synthetic hepcidin-25 in solution. The clone ultimatelyderived from hybridoma 5A3 was later renamed MAb 583; thus, data for MAb583 is shown in the 5A3 wells. Clones 4B1 and 5A4 although apparentlypositive in this screen both failed to produce functional anti-hepcidinantibodies when tested after further expansion, functionality, andisotype screening.

FIG. 5. Characteristics of MAbs 583, 1B1, and H32, including antigens,serum titers of immunized BALB/c mice, injection sites, injectionfrequency, tissue(s) used for hybridoma production, and success ratesthrough each round of screening leading to these isolated, functionalanti-hepcidin monoclonal antibodies.

FIG. 6. Overall success rates for functional anti-hepcidin-25 MAbsacross 8 MAb development campaigns. A total of 11,845 hybridomas werescreened for the discovery of MAbs H32, 583, and 1B1.

FIG. 7 Reducing SDS-PAGE analysis of purification of MAb 583 on aProtein A column. Crude preparations of diluted hybridoma supernatants,flow-through fractions collected during column washing, and highlypurified MAb 583 (Purification Lot 003) and 1B1 (Purification Lot 003)analyzed by Coomassie staining. Lane descriptions are provided to theright of the blot.

FIG. 8. ELISA analysis of neutralization of MAb 583 in solution byhepcidin-25. This solution-based screen tested the ability of 0.0, and0.1-8.0 ng synthetic hepcidin-25 (x axis) to block (neutralize) thebinding of 20 ng MAb 583 in solution. Synthetic hepcidin (0.1-8.0 ng)was added to 20 ng MAb 583 for two hours in binding buffer. Thehepcidin-25 treated MAb 583 solutions were added to duplicate wells withhepcidin-25 (200 ng/well) covalently bound to maleic anhydride activatedmicrowell plates. Binding to hepcidin-25 by MAb 583 was detected usingrabbit anti-mouse IgG (H+L) conjugated to HRP with TMB as the substrate.MAb 583 binding to bound hepcidin-25 is quantified by spectrophotometryafter addition of stop solution by measuring OD (optical density) at 450nm.

FIG. 9. Non-reducing tricine SDS-PAGE gel (left panel) stained withCoomassie and Western blot (right panel) of hepcidin-25, hepcidin-22,hepcidin-20, and protegrin (1.5 μg/lane) probed with MAb 583. Lanedescriptions are provided in the legend below the blot.

FIG. 10. Coomassie stained reducing SDS-PAGE and Western blots ofbinding activity of MAb 583 and MAb 1B1 against hepcidin-25,hepcidin-20, K18-biotin hepcidin-25, K24-biotin hepcidin-25, andNT-biotin hepcidin-25. Lane descriptions are provided in the legendbelow the blot.

FIG. 11. Biacore analysis of binding affinities of MAb 583 at 24 μg/ml,for K18-biotin hepcidin-25, NT-biotin hepcidin-25, and K24-biotinhepcidin-25 bound to a streptavidin coated Biacore chip. The data showhigh affinity and picomolar dissociation constants for MAb 583 forNT-biotin hepcidin-25, and approximately one log decreased affinityconstants for K18-biotin hepcidin-25 and K24-biotin hepcidin-25,respectively.

FIG. 12. Biacore analysis of binding affinities of MAb 583 at 5 μg/mlfor K18-biotin hepcidin-25 and NT-biotin hepcidin-25 bound to astreptavidin coated Biacore chip. There is little change in the slope ofthe Biacore trace over 1200 seconds (20 minutes) indicating a lowbinding dissociation constants for MAb 583 for K18-biotin hepcidin-25and NT-biotin hepcidin-25.

FIG. 13. Binding affinity results from Biacore experiments shown in FIG.12. The data show higher affinity and picomolar association (KA) andlower dissociation constants (KD) for MAb 583 for K18-biotin hepcidin-25than NT-biotin hepcidin-25 than when assessed at 24 μg/ml.

FIG. 14. Biacore data showing the binding affinity of MAb 1B1 at 24μg/ml for K18-biotin hepcidin-25, NT-biotin hepcidin-25, and K24-biotinhepcidin-25. These data show MAb 1B1 has strong binding affinity forK24-biotin hepcidin-25, lower affinity for K18-biotin hepcidin-25, andno affinity for NT-biotin hepcidin-25. This experiment was conducted for500 seconds and a no dissociation of 1B1 was observed or calculable bythe Biacore instrument after binding to K24-biotin hepcidin-25 andK18-biotin hepcidin-25 over 500 seconds.

FIG. 15. Biacore data showing binding affinity of MAb 1B1 at 24 μg/mlfor K24-biotin hepcidin-25. These data show strong affinity of 1B1 forK24-biotin hepcidin-25 and no dissociation of MAb 1B1 from K24-biotinhepcidin-25 over 500 seconds suggesting a low picomolar to femtamolardissociation constant for 1B1 for hepcidin-25 is possible.

FIG. 16. ELISA standard curve analysis of binding of hepcidin-25,hepcidin-22, and hepcidin-20 to MAb 583 antibody coated at 100 ng/ml perwell. The relative binding of the NT-biotin hepcidin-25 tracer (1ng/well) relative to hepcidin-25, hepcidin-22, and hepcidin-20 wasmeasured by ELISA. Four parameter logistical regression analysis wasconducted using GraphPad Prism software to produce the curves shown. Aright shift in the curve demonstrates consecutively lower affinity ofMAb 583 for hepcidin-22 and hepcidin-20, than MAb 583 has forhepcidin-25.

FIG. 17. ELISA analysis of binding to murine hepcidin-1 (mousehepcidin-25), protegrin, and hepcidin-(10-25) peptide to MAb 583compared to NT-biotin hepcidin-25. These data show that there is nobinding of MAb 583 to murine hepcidin-1, protegrin, or an oxidized andrefolded hepcidin-(10-25) peptide containing the proper cysteine bondsfor this region of hepcidin-25 at concentrations up to 2000 ng/ml.

FIG. 18. ELISA analysis of binding of hepcidin-(10-25) peptide to MAb1B1. The results indicate that MAb 1B1 has no binding affinity for anoxidized and refolded hepcidin-(10-25) peptide containing the propercysteine bonds for this region when compared to hepcidin-25. K18-biotinhepcidin-25 was used for detection. Note that there is no binding ofhepcidin-(10-25) peptide to MAb 1B1 at concentrations up to 2000 ng/ml.

FIG. 19. ELISA analysis of binding of hepcidin-(10-25) to MAb 1B1. Theresults indicate that MAb 1B1 has no binding affinity for an oxidizedand refolded hepcidin-(10-25) peptide containing the proper cysteinebonds for this region when compared to hepcidin-25. NT-biotinhepcidin-25 was used for detection. Note that there is no binding ofhepcidin-(10-25) to MAb 1B1 at concentrations up to 2000 ng/ml of thehepcidin-(10-25) peptide.

FIG. 20. Flow cytometry of Fpn-GFP cells treated with MAb 583. Cellswere induced overnight with ponasterone to induce expression of murineFpn-GFP. The next day, ponasterone was removed by washing, andhepcidin-25 and MAb 583 antibodies added for 24 hours. Hepcidin-25 wasused at 100 ng/ml concentration (37 nM). MAb 583 was added at 10-times,2-times or ⅓rd of hepcidin concentration (370 nM, 74 nM and 10 nM). Thecontrol MAb was a failed anti-hepcidin monoclonal antibody when screenedin vitro by ELISA and was used at the highest concentration (370 nM).Note that 10 nM MAb 583 neutralized completely 37 nM hepcidin-25 and itsbiological activity leading to degradation of FPN-GFP.

FIG. 21. Percent (%) change in FPN-GFP fluorescence in HEK cells treatedwith MAb 583 at concentrations from 10-370 nM in the presence of 37 nMhepcidin-25.

FIG. 22. Flow cytometry of Fpn-GFP cells treated with MAb 583. Cellswere induced overnight with ponasterone to induce expression of murineFpn-GFP. Next day, ponasterone was removed by washing, and hepcidin-25and MAb 583 antibodies added for 24 hours. Hepcidin-25 was used at 100ng/ml concentration (37 nM). MAb 583 was added at ⅓rd, ⅙th, 1/12th, and1/24th the molar concentration of hepcidin-25 in these cell based assaysof MAb 583 biological activity. The control MAb was a failedanti-hepcidin monoclonal antibody when screened in vitro by ELISA andwas used at the highest concentration (370 nM). Note that 2.5 nM MAb 583neutralized significantly (˜23% decrease) 37 nM hepcidin-25 and itbiological activity leading to degradation of FPN-GFP at 1/12^(th) ofthe molar ratio in vitro.

FIG. 23. Percent (%) change in FPN-GFP fluorescence in HEK cells treatedwith MAb 583 at concentrations from 0.62-10 nM in the presence of 37 nMhepcidin-25 as described in FIG. 22.

FIG. 24. Ferritin assay of Fpn-GFP cells treated with MAb 583. HEK cellswere induced overnight with ponasterone to induce expression of murineFpn-GFP and iron transport into the media. The next day, ponasterone wasremoved by washing, and hepcidin-25 and MAb 583 antibodies added for 24hours. Hepcidin-25 was used at 100 ng/ml concentration (37 nM). MAb 583was added at 10-times, 2-times or ⅓rd of hepcidin concentration (370 nM,74 nM and 10 nM). The control MAb was a failed anti-hepcidin monoclonalantibody when screened in vitro by ELISA and was used at the highestconcentration (370 nM). Note that 10 nM MAb 583 significantlyneutralized 37 nM hepcidin-25 and it biological activity leading todegradation of FPN-GFP and retention of intracellular ferritin boundiron. Proteins were extracted using RIPA buffer and intracellularferritin concentrations determined using ferritin ELISA (Ramco).

FIG. 25. Ferritin assay of Fpn-GFP cells treated with MAb 583. HEK cellswere induced overnight with ponasterone to induce expression of murineFpn-GFP and iron transport into the media. Next day, ponasterone wasremoved by washing, and hepcidin-25 and MAb 583 antibodies added for 24hours. Hepcidin-25 was used at 100 ng/ml concentration (37 nM) and MAb583 antibody at ⅓rd, ⅙th, 1/12th, and 1/24th the molar concentration ofhepcidin-25 in these cell based assay of MAb 583 biological activity.The control MAb (sham MAb) was a failed anti-hepcidin monoclonalantibody when screened in vitro by ELISA and was used at the highestconcentration (370 nM). Note that 2.5-5 nM 583 significantly neutralized37 nM hepcidin-25 and it biological activity leading to degradation ofFPN-GFP and retention of intracellular ferritin bound iron. Proteinswere extracted using RIPA buffer and intracellular ferritinconcentrations determined using ferritin ELISA (Ramco).

FIG. 26. Percent (%) change of intracellular ferritin concentration inHEK cells treated with MAb 583 at concentrations from 0.62-10 nM in thepresence of 37 nM hepcidin-25 as described in FIG. 25.

FIG. 27. Effect of injection of MAb 583 and human hepcidin-25 in vivo onserum iron concentration in male C57Bl/6 mice. Five groups of mice(n=8/group) were injected intraperitoneally with either PBS (group 1,H-PBS and group 5, PBS), 1 mg MAb 583 (group 2, H-Mab), 0.5 mg MAb 583(group 3, H-2Mab), or 0.5 mg of control Mab (group 4, H-sham Mab). Thefollowing day all mice in group 3 received an additional 0.5 mg of MAb583 and 24 hours later groups 1-4 received a single injection of 50 μgof human hepcidin-25 and group 5 received PBS. All mice were sacrificed2 hours later and serum iron was measured. Statistical analysis (seeFIG. 28 for details) indicated a significant difference between PBS andPBS plus hepcidin-25 (H-PBS, P=0.001), and between PBS plus hepcidin-25(H-PBS) and mice that received two doses of 0.5 mg of MAb 583 andhepcidin-25 (H-2Mab, P=0.004).

FIG. 28. Descriptive statistics (mean, standard deviation, SEM) andresults from one way ANOVA of serum iron concentrations of five groupsof male C57BL/6 mice from the in vivo study of MAb 583 shown in FIG. 27.Multiple Comparisons versus Control Group (Holm-Sidak method) with anoverall significance level of P=0.05 gave comparison-dependentunadjusted P values ranging from 0.001 to 0.253.

FIG. 29. Comparison of the MAb 583 chimera to murine MAb 583 for bindingto a Hepcidin-25 coated ELISA plate. 100 ng hepcidin-25 was covalentlybound to wells of a maleic anhydride activated 96 well microplate.Increasing amounts of MAb 583 chimera (BAP070-01; 3650 ng/ml; opensquares) and murine MAb 583 (positive control MAb; open triangles) wereadded to microwell plate and allowed to bind for one hour. Binding ofMAb 583 chimera was detected by rabbit anti-human IgG₁ (H+L) HRP. Boundmurine MAb 583 antibody was detected with anti-mouse IgG₁ (H+L)conjugated with HRP. The reactions were stopped with 1N HCl at 5 minutesafter TMB was added to the wells and read immediately. Binding wasquantified as OD on a spectrophotometer at 450 nm after addition of stopsolution. X axis: Antibody concentration in ng/ml; and Y axis: OD 450 nmvalues.

FIG. 30. MAb 583 chimera binding to a hepcidin-25 coated ELISA plate.100 ng hepcidin-25 was covalently bound to wells of a maleic anhydrideactivated 96 well microplate. Increasing amounts of MAb 583 chimera(BAP070-01; 3650 ng/ml; filled circles) and supernatant from cellstransfected with empty vector (BAP070; filled squares) were added tomicrowell plate and allowed to bind for 2 hours. Binding of MAb 583chimera was detected by rabbit anti-human IgG1 (H+L) HRP with TMB assubstrate. Binding was quantified on a spectrophotometer at 450 nm afteraddition of stop solution.

FIG. 31. Hepcidin-25 standard curve produced using the BAP070-01 MAb 583chimera. Wells on microwell plate were coated with 150 ng/ml Protein Gand blocked. The Mab 583 chimera (BAP070-01) was added to the wells at150 ng/well and allowed to bind for one hours. Known concentrations ofsynthetic hepcidin-25 was added to assay buffer containing NT-biotinhepcidin-25 (ing/well), mixed, and added to 8 duplicate wells andallowed to compete for two hours. The wells were washed and SA-HRP withTMB substrate was used to detect binding of the NT-biotin hepcidin-25tracer. Binding was quantified on a spectrophotometer at 450 nm afteraddition of stop solution. The standard curve was generated usingGraphpad Prism software (San Diego, Calif.) using a 4-parameterlogistical regression.

FIG. 32. Binding of MAb 583 chimera to NT-biotin hepcidin-25 onneutravidin coated microwell plates. Wells on microwell plate werecoated with 150 ng/ml neutravidin and blocked. The NT-biotin hepcidin-25tracer was added to wells at ing/well and allowed to bind for one hour.The Mab 583 chimera (BAP070-01) was added to the wells at 150 ng/wellalong with synthetic hepcidin-25 at known concentrations and allowed tocompete for binding to NT-biotin hepcidin-25 for one hour. Binding ofthe Mab 583 chimera was detected using rabbit anti-human IgG1 (H+L) HRPwith TMB as substrate. Binding was quantified by spectropscopy at 450 nmafter addition of stop solution. The points represent the two duplicates(filled diamonds and squares) and the mean (filled triangles).

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present application, there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook et al, “Molecular Cloning:A Laboratory Manual” (1989); “Current Protocols in Molecular Biology”Volumes I-III [Ausubel, R. M., ed. (1994)]; “Cell Biology: A LaboratoryHandbook” Volumes I-III [J. E. Celis, ed. (1994))]; “Current Protocolsin Immunology” Volumes I-III [Coligan, J. E., ed. (1994)];“Oligonucleotide Synthesis” (M. J. Gait ed. 1984); “Nucleic AcidHybridization” [B. D. Hames & S. J. Higgins eds. (1985)]; “TranscriptionAnd Translation” [B. D. Hames & S. J. Higgins, eds. (1984)]; “AnimalCell Culture” [R. I. Freshney, ed. (1986)]; “Immobilized Cells AndEnzymes” [IRL Press, (1986)]; B. Perbal, “A Practical Guide To MolecularCloning” (1984), each of which is specifically incorporated herein byreference in its entirety.

Antibody Terminology

As used herein, the term “antibody” refers to an immunoglobulin (Ig)whether natural or partly or wholly synthetically produced. The termalso covers any polypeptide or protein having a binding domain which is,or is homologous to, an antigen-binding domain. The term furtherincludes “antigen-binding fragments” and other interchangeable terms forsimilar binding fragments such as described below. Complementaritydetermining region (CDR) grafted antibodies and other humanizedantibodies (including CDR modifications and framework regionmodifications) are also contemplated by this term.

Native antibodies and native immunoglobulins are usuallyheterotetrameric glycoproteins of about 150,000 Daltons, composed of twoidentical light (L) chains and two identical heavy (H) chains. Eachlight chain is typically linked to a heavy chain by one covalentdisulfide bond, while the number of disulfide linkages varies among theheavy chains of different immunoglobulin isotypes. Each heavy and lightchain also has regularly spaced intrachain disulfide bridges. Each heavychain has at one end a variable domain (“V_(H)”) followed by a number ofconstant domains (“C_(H)”). Each light chain has a variable domain atone end (“V_(L)”) and a constant domain (“C_(L)”) at its other end; theconstant domain of the light chain is aligned with the first constantdomain of the heavy chain, and the light-chain variable domain isaligned with the variable domain of the heavy chain. Particular aminoacid residues are believed to form an interface between the light- andheavy-chain variable domains.

The terms “synthetic polynucleotide,” “synthetic gene” or “syntheticpolypeptide,” as used herein, mean that the corresponding polynucleotidesequence or portion thereof, or amino acid sequence or portion thereof,is derived, from a sequence that has been designed, or synthesized denovo, or modified, compared to an equivalent naturally-occurringsequence. Synthetic polynucleotides (antibodies or antigen bindingfragments) or synthetic genes can be prepared by methods known in theart, including but not limited to, the chemical synthesis of nucleicacid or amino acid sequences. Synthetic genes are typically differentfrom naturally-occurring genes, either at the amino acid, orpolynucleotide level, (or both) and are typically located within thecontext of synthetic expression control sequences. For example,synthetic gene sequences can include amino acid, or polynucleotide,sequences that have been changed, for example, by the replacement,deletion, or addition, of one or more, amino acids, or nucleotides,thereby providing an antibody amino acid sequence, or a polynucleotidecoding sequence that is different from the source sequence. Syntheticgene polynucleotide sequences, may not necessarily encode proteins withdifferent amino acids, compared to the natural gene; for example, theycan also encompass synthetic polynucleotide sequences that incorporatedifferent codons but which encode the same amino acid (i.e., thenucleotide changes represent silent mutations at the amino acid level).

With respect to antibodies, the term “variable domain” refers to thevariable domains of antibodies that are used in the binding andspecificity of each particular antibody for its particular antigen.However, the variability is not evenly distributed throughout thevariable domains of antibodies. Rather, it is concentrated in threesegments called hypervariable regions (also known as CDRs) in both thelight chain and the heavy chain variable domains. More highly conservedportions of variable domains are called the “framework regions” or“FRs.” The variable domains of unmodified heavy and light chains eachcontain four FRs (FR1, FR2, FR3 and FR4), largely adopting a β-sheetconfiguration interspersed with three CDRs which form loops connectingand, in some cases, part of the β-sheet structure. The CDRs in eachchain are held together in close proximity by the FRs and, with the CDRsfrom the other chain, contribute to the formation of the antigen-bindingsite of antibodies (see Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991), pages 647-669).

The terms “hypervariable region” and “CDR” when used herein, refer tothe amino acid residues of an antibody which are responsible forantigen-binding. The CDRs comprise amino acid residues from threesequence regions which bind in a complementary manner to an antigen andare known as CDR1, CDR2, and CDR3 for each of the V_(H) and V_(L)chains. In the light chain variable domain, the CDRs typicallycorrespond to approximately residues 24-34 (CDRL1), 50-56 (CDRL2) and89-97 (CDRL3), and in the heavy chain variable domain the CDRs typicallycorrespond to approximately residues 31-35 (CDRH1), 50-65 (CDRH2) and95-102 (CDRH3) according to Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991)). It is understood that theCDRs of different antibodies may contain insertions, thus the amino acidnumbering may differ. The Kabat numbering system accounts for suchinsertions with a numbering scheme that utilizes letters attached tospecific residues (e.g., 27A, 27B, 27C, 27D, 27E, and 27F of CDRL1 inthe light chain) to reflect any insertions in the numberings betweendifferent antibodies. Alternatively, in the light chain variable domain,the CDRs typically correspond to approximately residues 26-32 (CDRL1),50-52 (CDRL2) and 91-96 (CDRL3), and in the heavy chain variable domain,the CDRs typically correspond to approximately residues 26-32 (CDRH1),53-55 (CDRH2) and 96-101 (CDRH3) according to Chothia and Lesk, J. Mol.Biol., 196: 901-917 (1987)).

As used herein, “framework region” or “FR” refers to framework aminoacid residues that form a part of the antigen binding pocket or groove.In some embodiments, the framework residues form a loop that is a partof the antigen binding pocket or groove and the amino acids residues inthe loop may or may not contact the antigen. Framework regions generallycomprise the regions between the CDRs. In the light chain variabledomain, the FRs typically correspond to approximately residues 0-23(FRL1), 35-49 (FRL2), 57-88 (FRL3), and 98-109 and in the heavy chainvariable domain the FRs typically correspond to approximately residues0-30 (FRH1), 36-49 (FRH2), 66-94 (FRH3), and 103-133 according to Kabatet al., Sequences of Proteins of Immunological Interest, 5th Ed. PublicHealth Service, National Institutes of Health, Bethesda, Md. (1991)). Asdiscussed above with the Kabat numbering for the light chain, the heavychain too accounts for insertions in a similar manner (e.g., 35A, 35B ofCDRH1 in the heavy chain). Alternatively, in the light chain variabledomain, the FRs typically correspond to approximately residues 0-25(FRL1), 33-49 (FRL2) 53-90 (FRL3), and 97-109 (FRL4), and in the heavychain variable domain, the FRs typically correspond to approximatelyresidues 0-25 (FRH1), 33-52 (FRH2), 56-95 (FRH3), and 102-113 (FRH4)according to Chothia and Lesk, J. Mol. Biol., 196: 901-917 (1987)).

The loop amino acids of a FR can be assessed and determined byinspection of the three-dimensional structure of an antibody heavy chainand/or antibody light chain. The three-dimensional structure can beanalyzed for solvent accessible amino acid positions as such positionsare likely to form a loop and/or provide antigen contact in an antibodyvariable domain. Some of the solvent accessible positions can tolerateamino acid sequence diversity and others (e.g., structural positions)are, generally, less diversified. The three dimensional structure of theantibody variable domain can be derived from a crystal structure orprotein modeling.

Constant domains (Fc) of antibodies are not involved directly in bindingan antibody to an antigen but, rather, exhibit various effectorfunctions, such as participation of the antibody in antibody-dependentcellular toxicity via interactions with, for example, Fc receptors(FcR). Fc domains can also increase bioavailability of an antibody incirculation following administration to a subject.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, andIgM, and several of these can be further divided into subclasses(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Theheavy-chain constant domains (Fc) that correspond to the differentclasses of immunoglobulins are called α, δ, ε, γ, and μ, respectively.The subunit structures and three-dimensional configurations of differentclasses of immunoglobulins are well known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa or (“κ”) and lambda or (“λ”), based on the amino acid sequences oftheir constant domains.

The terms “antigen-binding portion of an antibody,” “antigen-bindingfragment,” “antigen-binding domain,” “antibody fragment” or a“functional fragment of an antibody” are used interchangeably herein torefer to one or more fragments of an antibody that retain the ability tospecifically bind to an antigen. Non-limiting examples of antibodyfragments included within such terms include, but are not limited to,(i) a Fab fragment, a monovalent fragment consisting of the V_(L),V_(H), C_(L) and C_(H1) domains; (ii) a F(ab′)₂ fragment, a bivalentfragment containing two Fab fragments linked by a disulfide bridge atthe hinge region; (iii) a Fd fragment consisting of the V_(H) and C_(H1)domains; (iv) a Fv fragment containing the V_(L) and V_(H) domains of asingle arm of an antibody, (v) a dAb fragment (Ward et al., (1989)Nature 341:544 546), which containing a V_(H) domain; and (vi) anisolated CDR. Additionally included in this definition are “one-half”antibodies comprising a single heavy chain and a single light chain.Other forms of single chain antibodies, such as diabodies are alsoencompassed herein.

“F(ab′)₂” and “Fab′” moieties can be produced by treating an Ig with aprotease such as pepsin and papain, and include antibody fragmentsgenerated by digesting immunoglobulin near the disulfide bonds existingbetween the hinge regions in each of the two heavy chains. For example,papain cleaves IgG upstream of the disulfide bonds existing between thehinge regions in each of the two heavy chains to generate two homologousantibody fragments in which an light chain composed of V_(L) and C_(L)(light chain constant region), and a heavy chain fragment composed ofV_(H) and C_(Hγ1) (γ1) region in the constant region of the heavy chain)are connected at their C terminal regions through a disulfide bond. Eachof these two homologous antibody fragments is called Fab′. Pepsin alsocleaves IgG downstream of the disulfide bonds existing between the hingeregions in each of the two heavy chains to generate an antibody fragmentslightly larger than the fragment in which the two above-mentioned Fab′are connected at the hinge region. This antibody fragment is calledF(ab′)₂.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (C_(H)1) of the heavy chain. Fab′fragments differ from Fab fragments by the addition of a few residues atthe carboxyl terminus of the heavy chain C_(H)1 domain including one ormore cysteine(s) from the antibody hinge region. Fab′-SH is thedesignation herein for Fab′ in which the cysteine residue(s) of theconstant domains bear a free thiol group. F(ab′)₂ antibody fragmentsoriginally were produced as pairs of Fab′ fragments which have hingecysteines between them. Other chemical couplings of antibody fragmentsare also known.

“Fv” refers to an antibody fragment which contains a completeantigen-recognition and antigen-binding site. This region consists of adimer of one heavy chain and one light chain variable domain in tight,non-covalent or covalent association (disulfide linked Fv's have beendescribed in the art, Reiter et al. (1996) Nature Biotechnology14:1239-1245). It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the V_(H)-V_(L) dimer. Collectively, a combination of one ormore of the CDRs from each of the V_(H) and V_(L) chains conferantigen-binding specificity to the antibody. For example, it would beunderstood that, for example, the CDRH3 and CDRL3 could be sufficient toconfer antigen-binding specificity to an antibody when transferred toV_(H) and V_(L) chains of a recipient antibody or antigen-bindingfragment thereof and this combination of CDRs can be tested for binding,affinity, etc. using any of the techniques described herein. Even asingle variable domain (or half of an Fv comprising only three CDRsspecific for an antigen) has the ability to recognize and bind antigen,although likely at a lower affinity than when combined with a secondvariable domain. Furthermore, although the two domains of a Fv fragment(V_(L) and V_(H)), are coded for by separate genes, they can be joinedusing recombinant methods by a synthetic linker that enables them to bemade as a single protein chain in which the V_(L) and V_(H) regions pairto form monovalent molecules (known as single chain Fv (scFv); Bird etal. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad.Sci. USA 85:5879-5883; and Osbourn et al. (1998) Nat. Biotechnol.16:778). Such scFvs are also intended to be encompassed within the term“antigen-binding portion” of an antibody. Any V_(H) and V_(L) sequencesof specific scFv can be linked to an Fc region cDNA or genomicsequences, in order to generate expression vectors encoding complete Ig(e.g., IgG) molecules or other isotypes. V_(H) and V_(L) can also beused in the generation of Fab, Fv or other fragments of Igs using eitherprotein chemistry or recombinant DNA technology.

“Single-chain Fv” or “sFv” antibody fragments comprise the V_(H) andV_(L) domains of an antibody, wherein these domains are present in asingle polypeptide chain. In some embodiments, the Fv polypeptidefurther comprises a polypeptide linker between the V_(H) and V_(L)domains which enables the sFv to form the desired structure for antigenbinding. For a review of sFvs, see, e.g., Pluckthun in The Pharmacologyof Monoclonal Antibodies, Vol. 113, Rosenburg and Moore eds.Springer-Verlag, New York, pp. 269-315 (1994).

The term “AVIMER™” refers to a class of therapeutic proteins of humanorigin, which are unrelated to antibodies and antibody fragments, andare composed of several modular and reusable binding domains, referredto as A-domains (also referred to as class A module, complement typerepeat, or LDL-receptor class A domain). They were developed from humanextracellular receptor domains by in vitro exon shuffling and phagedisplay (Silverman et al., 2005, Nat. Biotechnol. 23:1493-1494;Silverman et al., 2006, Nat. Biotechnol. 24:220). The resulting proteinscan contain multiple independent binding domains that can exhibitimproved affinity (in some cases, sub-nanomolar) and specificitycompared with single-epitope binding proteins. See, for example, U.S.Patent Application Publ. Nos. 2005/0221384, 2005/0164301, 2005/0053973and 2005/0089932, 2005/0048512, and 2004/0175756, each of which ishereby incorporated by reference herein in its entirety.

Each of the known 217 human A-domains comprises ˜35 amino acids (˜4kDa); and these domains are separated by linkers that average five aminoacids in length. Native A-domains fold quickly and efficiently to auniform, stable structure mediated primarily by calcium binding anddisulfide formation. A conserved scaffold motif of only 12 amino acidsis required for this common structure. The end result is a singleprotein chain containing multiple domains, each of which represents aseparate function. Each domain of the proteins binds independently andthe energetic contributions of each domain are additive. These proteinswere called “AVIMERs™” from avidity multimers.

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy chain variabledomain (V_(H)) connected to a light chain variable domain (V_(L)) in thesame polypeptide chain (V_(H)-V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA 90:6444 6448 (1993).

Antigen-binding polypeptides also include heavy chain dimers such as,for example, antibodies from camelids and sharks. Camelid and sharkantibodies comprise a homodimeric pair of two chains of V-like andC-like domains (neither has a light chain). Since the V_(H) region of aheavy chain dimer IgG in a camelid does not have to make hydrophobicinteractions with a light chain, the region in the heavy chain thatnormally contacts a light chain is changed to hydrophilic amino acidresidues in a camelid. V_(H) domains of heavy-chain dimer IgGs arecalled V_(HH) domains. Shark Ig-NARs comprise a homodimer of onevariable domain (termed a V-NAR domain) and five C-like constant domains(C-NAR domains). In camelids, the diversity of antibody repertoire isdetermined by the CDRs 1, 2, and 3 in the V_(H) or V_(HH) regions. TheCDR3 in the camel V_(HH) region is characterized by its relatively longlength, averaging 16 amino acids (Muyldermans et al., 1994, ProteinEngineering 7(9): 1129). This is in contrast to CDR3 regions ofantibodies of many other species. For example, the CDR3 of mouse V_(H)has an average of 9 amino acids. Libraries of camelid-derived antibodyvariable regions, which maintain the in vivo diversity of the variableregions of a camelid, can be made by, for example, the methods disclosedin U.S. Patent Application Ser. No. 20050037421.

“Humanized” forms of non-human (e.g., murine) antibodies includechimeric antibodies which contain minimal sequence derived from anon-human Ig. For the most part, humanized antibodies are human IgGs(recipient antibody) in which one or more of the CDRs of the recipientare replaced by CDRs from a non-human species antibody (donor antibody)such as mouse, rat, rabbit or non-human primate having the desiredspecificity, affinity and binding function. In some instances, one ormore FR amino acid residues of the human Ig are replaced bycorresponding non-human amino acid residues. Furthermore, humanizedantibodies can contain residues which are not found in the recipientantibody or in the donor antibody. These modifications can be made torefine antibody performance, if needed. A humanized antibody cancomprise substantially all of at least one and, in some cases two,variable domains, in which all or substantially all of the hypervariableregions correspond to those of a non-human immunoglobulin and all, orsubstantially all, of the FRs are those of a human immunoglobulinsequence. The humanized antibody optionally can also include at least aportion of an immunoglobulin constant region (Fc), typically that of ahuman immunoglobulin. For details, see Jones et al., Nature 321: 522-525(1986); Reichmann et al., Nature 332: 323-329 (1988); and Presta, Curr.Op. Struct. Biol. 2: 593-596 (1992).

A humanized antibody also includes antibodies in which part, or all ofthe CDRs of the heavy and light chain are derived from a non-humanmonoclonal antibody, substantially all the remaining portions of thevariable regions are derived from human variable region (both heavy andlight chain), and the constant regions are derived from a human constantregion. In one embodiment, the CDR1, CDR2 and CDR3 regions of the heavyand light chains are derived from a non-human antibody. In yet anotherembodiment, at least one CDR (e.g., a CDR3) of the heavy and lightchains is derived from a non-human antibody. Various combinations ofCDR1, CDR2, and CDR3 can be derived from a non-human antibody and arecontemplated herein. In one non-limiting example, one or more of theCDR1, CDR2 and CDR3 regions of each of the heavy and light chains arederived from the sequences provided herein.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations, which can includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. The modifier “monoclonal” indicates the character of theantibody as being obtained from a substantially homogeneous populationof antibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, monoclonal antibodiescan be made by the hybridoma method first described by Kohler et al.,Nature 256:495 (1975), or can be made by recombinant DNA methods (see,e.g., U.S. Pat. No. 4,816,567). In certain embodiments, the monoclonalantibodies can be isolated from phage antibody libraries using thetechniques described in Clackson et al., Nature 352:624-628 (1991) andMarks et al., J. Mol. Biol. 222:581-597 (1991), for example.

Antibodies can be isolated and purified from the culture supernatant orascites mentioned above by saturated ammonium sulfate precipitation,euglobulin precipitation method, caproic acid method, caprylic acidmethod, ion exchange chromatography (DEAE or DE52), or affinitychromatography using anti-Ig column or a protein A, G or L column suchas described in more detail below.

Exemplary antibodies for use in the compositions and methods describedherein are intact immunoglobulin molecules, such as, for example, ahumanized antibody or those portions of a humanized Ig molecule thatcontain the antigen binding site (i.e., paratope) or a single heavychain and a single light chain, including those portions known in theart as Fab, Fab′, F(ab)′, F(ab′)₂, Fd, scFv, a variable heavy domain, avariable light domain, a variable NAR domain, bi-specific scFv, abi-specific Fab₂, a tri-specific Fab₃ and a single chain bindingpolypeptides and others also referred to as antigen-binding fragments.When constructing an immunoglobulin molecule or fragments thereof,variable regions or portions thereof may be fused to, connected to, orotherwise joined to one or more constant regions or portions thereof toproduce any of the antibodies or fragments thereof described herein.This may be accomplished in a variety of ways known in the art,including but not limited to, molecular cloning techniques or directsynthesis of the nucleic acids encoding the molecules. Exemplarynon-limiting methods of constructing these molecules can also be foundin the examples described herein.

Methods for making bispecific or other multispecific antibodies areknown in the art and include chemical cross-linking, use of leucinezippers (Kostelny et al., J. Immunol. 148:1547-1553, 1992); diabodytechnology (Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-48,1993); scFv dimers [Gruber et al., J. Immunol. 152: 5368, 1994], linearantibodies (Zapata et al., Protein Eng. 8:1057-62, 1995); and chelatingrecombinant antibodies (Neri et al., J Mol Biol. 246:367-73, 1995).

“Linear antibodies” comprise a pair of tandem Fd segments(V_(H)-C_(H)1-V_(H)-C_(H)1) which form a pair of antigen bindingregions. Linear antibodies can be bispecific or monospecific (Zapata etal. Protein Eng. 8:1057-62 (1995)).

Additionally, the anti-hepcidin antibodies disclosed herein can also beconstructed to fold into multivalent forms, which may improve bindingaffinity, specificity and/or increased half-life in blood. Multivalentforms of anti-hepcidin antibodies can be prepared by techniques known inthe art.

Bispecific or multispecific antibodies include cross-linked or“heteroconjugate” antibodies. For example, one of the antibodies in theheteroconjugate can be coupled to avidin, the other to biotin.Heteroconjugate antibodies may be made using any convenientcross-linking methods. Suitable cross-linking agents are well known inthe art, and are disclosed in U.S. Pat. No. 4,676,980, along with anumber of cross-linking techniques. Another method is designed to maketetramers by adding a streptavidin-coding sequence at the C-terminus ofthe scFv. Streptavidin is composed of four subunits, so when thescFv-streptavidin is folded, four subunits associate to form a tetramer(Kipriyanov et al., Hum Antibodies Hybridomas 6(3): 93-101 (1995), thedisclosure of which is incorporated herein by reference in itsentirety).

According to another approach for making bispecific antibodies, theinterface between a pair of antibody molecules can be engineered tomaximize the percentage of heterodimers which are recovered fromrecombinant cell culture. One interface comprises at least a part of theC_(H)3 domain of an antibody constant domain. In this method, one ormore small amino acid side chains from the interface of the firstantibody molecule are replaced with larger side chains (e.g., tyrosineor tryptophan). Compensatory “cavities” of identical or similar size tothe large side chain(s) are created on the interface of the secondantibody molecule by replacing large amino acid side chains with smallerones (e.g., alanine or threonine). This provides a mechanism forincreasing the yield of the heterodimer over other unwanted end-productssuch as homodimers. See WO 96/27011 published Sep. 6, 1996.

Techniques for generating bispecific or multispecific antibodies fromantibody fragments are conventionally known in the art. For example,bispecific or trispecific antibodies can be prepared using chemicallinkage. Brennan et al., Science 229:81 (1985) describe a procedurewherein intact antibodies are proteolytically cleaved to generateF(ab′)₂ fragments. These fragments are reduced in the presence of thedithiol complexing agent sodium arsenite to stabilize vicinal dithiolsand prevent intermolecular disulfide formation. The Fab′ fragmentsgenerated are then converted to thionitrobenzoate (TNB) derivatives. Oneof the Fab′-TNB derivatives is then reconverted to the Fab′-thiol byreduction with mercaptoethylamine and is mixed with an equimolar amountof the other Fab′-TNB derivative to form the bispecific antibody. Thebispecific antibodies produced can be used as agents for the selectiveimmobilization of enzymes. Better et al., Science 240: 1041-1043 (1988)disclose secretion of functional antibody fragments from bacteria (see,e.g., Better et al., Skerra et al. Science 240: 1038-1041 (1988)). Forexample, Fab′-SH fragments can be directly recovered from E. coli andchemically coupled to form bispecific antibodies (Carter et al.,Bio/Technology 10:163-167 (1992); Shalaby et al., J. Exp. Med.175:217-225 (1992)).

Various techniques for making and isolating bispecific or multispecificantibody fragments directly from recombinant cell culture have areconventionally known in the art. For example, bispecific antibodies havebeen produced using leucine zippers, e.g., GCN4. (See generally Kostelnyet al., J. Immunol. 148(5):1547-1553 (1992).) The leucine zipperpeptides from the Fos and Jun proteins were linked to the Fab′ portionsof two different antibodies by gene fusion. The antibody homodimers werereduced at the hinge region to form monomers and then re-oxidized toform the antibody heterodimers. This method can also be utilized for theproduction of antibody homodimers.

As used herein, a “minibody” refers to a scFv fused to CH₃ via a peptidelinker (hingeless) or via an IgG hinge has been described in Olafsen, etal., Protein Eng Des Sel. April 2004; 17(4):315-23.

As used herein, a “maxibody” refers to a bivalent scFv covalentlyattached to the Fc region of an immunoglobulin, see, for example,Fredericks et al., Protein Engineering, Design & Selection, 17:95-106(2004) and Powers et al., Journal of Immunological Methods, 251:123-135(2001).

As used herein, an “intrabody” refers to a single chain antibody whichdemonstrates intracellular expression and can manipulate intracellularprotein function (Biocca, et al., EMBO J. 9:101-108, 1990; Colby et al.,Proc Natl Acad Sci USA. 101:17616-21, 2004). Intrabodies, which comprisecell signal sequences which retain the antibody construct inintracellular regions, may be produced as described in Mhashilkar etal., (EMBO J 14:1542-51, 1995) and Wheeler et al. (FASEB J. 17:1733-5.2003). Transbodies are cell-permeable antibodies in which a proteintransduction domains (PTD) is fused with single chain variable fragment(scFv) antibodies Heng et al., (Med Hypotheses. 64:1105-8, 2005).

Additionally contemplated herein are antibodies that are SMIPs orbinding domain immunoglobulin fusion proteins specific for targetprotein. These constructs are single-chain polypeptides comprisingantigen binding domains fused to immunoglobulin domains necessary tocarry out antibody effector functions. See e.g., WO 03/041600, U.S.Patent publication 20030133939 and US Patent Publication 20030118592,which are hereby incorporated by reference.

Humanization of antibodies and antigen-binding fragments thereof, can beaccomplished via a variety of methods known in the art and describedherein. Similarly, production of humanized antibodies can also beaccomplished via methods known in the art and described herein.

In one exemplary embodiment, the application contemplates a single chainbinding polypeptide having a heavy chain variable region, and/or a lightchain variable region which binds an epitope described herein and has,optionally, an immunoglobulin Fc region. Such a molecule is a singlechain variable fragment (scFv) optionally having effector function orincreased half-life through the presence of the immunoglobulin Fcregion. Methods of preparing single chain binding polypeptides are knownin the art (e.g., U.S. Patent Application No. 2005/0238646).

The terms “germline gene segments” or “germline sequences” refer to thegenes from the germline (the haploid gametes and those diploid cellsfrom which they are formed). The germline DNA contains multiple genesegments that encode a single Ig heavy or light chain. These genesegments are carried in the germ cells but cannot be transcribed andtranslated into heavy and light chains until they are arranged intofunctional genes. During B-cell differentiation in the bone marrow,these gene segments are randomly shuffled by a dynamic genetic systemcapable of generating more than 10⁸ specificities. Most of these genesegments are published and collected by the germline database.

Binding affinity and/or avidity of antibodies or antigen-bindingfragments thereof may be improved by modifying framework regions.Methods for modifications of framework regions are known in the art andare contemplated herein. Selection of one or more relevant frameworkamino acid positions to altered depends on a variety of criteria. Onecriterion for selecting relevant framework amino acids to change can bethe relative differences in amino acid framework residues between thedonor and acceptor molecules. Selection of relevant framework positionsto alter using this approach has the advantage of avoiding anysubjective bias in residue determination or any bias in CDR bindingaffinity contribution by the residue.

As used herein, “immunoreactive” refers to antibodies or antigen-bindingfragments thereof that are specific to a sequence of amino acid residues(“binding site” or “epitope”), yet if are cross-reactive to otherpeptides/proteins, are not toxic at the levels at which they areformulated for administration to human use. The term “binding” refers toa direct association between two molecules, due to, for example,covalent, electrostatic, hydrophobic, and ionic and/or hydrogen-bondinteractions under physiological conditions, and including interactionssuch as salt bridges and water bridges and any other conventionalbinding means. The term “preferentially binds” means that the bindingagent binds to the binding site with greater affinity than it bindsunrelated amino acid sequences. Preferably such affinity is at least1-fold greater, at least 2-fold greater, at least 3-fold greater, atleast 4-fold greater, at least 5-fold greater, at least 6-fold greater,at least 7-fold greater, at least 8-fold greater, at least 9-foldgreater, 10-fold greater, at least 20-fold greater, at least 30-foldgreater, at least 40-fold greater, at least 50-fold greater, at least60-fold greater, at least 70-fold greater, at least 80-fold greater, atleast 90-fold greater, at least 100-fold greater, or at least 1000-foldgreater than the affinity of the binding agent for unrelated amino acidsequences. The terms “immunoreactive” and “preferentially binds” areused interchangeably herein.

As used herein, the term “affinity” refers to the equilibrium constantfor the reversible binding of two agents and is expressed as Kd. In oneembodiment, the antibodies, or antigen-binding fragments thereof exhibitdesirable characteristics such as binding affinity as measured by K_(D)(equilibrium dissociation constant) for hepcidin in the range of 1×10⁻⁶M or less, or ranging down to 10⁻¹⁶ M or lower, (e.g., about 10⁻⁷, 10⁻⁸,10⁻⁹, 10⁻¹⁰, 10⁻¹¹, 10⁻¹², 10⁻¹³, 10⁻¹⁴, 10⁻¹⁵, 10⁻¹⁶ M or less). Theequilibrium dissociation constant can be determined in solutionequilibrium assay using BIAcore and/or KinExA. As used herein, the term“avidity” refers to the resistance of a complex of two or more agents todissociation after dilution. Apparent affinities can be determined bymethods such as an enzyme linked immunosorbent assay (ELISA) or anyother technique familiar to one of skill in the art. Avidities can bedetermined by methods such as a Scatchard analysis or any othertechnique familiar to one of skill in the art.

“Epitope” refers to that portion of an antigen or other macromoleculecapable of forming a binding interaction with the variable regionbinding pocket of an antibody. Such binding interactions can bemanifested as an intermolecular contact with one or more amino acidresidues of one or more CDRs. Antigen binding can involve, for example,a CDR3 or a CDR3 pair or, in some cases, interactions of up to all sixCDRs of the V_(H) and V_(L) chains. An epitope can be a linear peptidesequence (i.e., “continuous”) or can be composed of noncontiguous aminoacid sequences (i.e., “conformational” or “discontinuous”). An antibodycan recognize one or more amino acid sequences; therefore an epitope candefine more than one distinct amino acid sequence. Epitopes recognizedby antibodies can be determined by peptide mapping and sequence analysistechniques well known to one of skill in the art. Binding interactionsare manifested as intermolecular contacts with one or more amino acidresidues of a CDR.

The term “specific” refers to a situation in which an antibody will notshow any significant binding to molecules other than the antigencontaining the epitope recognized by the antibody. The term is alsoapplicable where for example, an antigen binding domain is specific fora particular epitope which is carried by a number of antigens, in whichcase the antibody or antigen-binding fragment thereof carrying theantigen binding domain will be able to bind to the various antigenscarrying the epitope. The terms “preferentially binds” or “specificallybinds” mean that the antibodies or fragments thereof bind to an epitopewith greater affinity than it binds unrelated amino acid sequences, and,if cross-reactive to other polypeptides containing the epitope, are nottoxic at the levels at which they are formulated for administration tohuman use. In one aspect, such affinity is at least 1-fold greater, atleast 2-fold greater, at least 3-fold greater, at least 4-fold greater,at least 5-fold greater, at least 6-fold greater, at least 7-foldgreater, at least 8-fold greater, at least 9-fold greater, 10-foldgreater, at least 20-fold greater, at least 30-fold greater, at least40-fold greater, at least 50-fold greater, at least 60-fold greater, atleast 70-fold greater, at least 80-fold greater, at least 90-foldgreater, at least 100-fold greater, or at least 1000-fold greater thanthe affinity of the antibody or fragment thereof for unrelated aminoacid sequences. The terms “immunoreactive,” “binds,” “preferentiallybinds” and “specifically binds” are used interchangeably herein. Theterm “binding” refers to a direct association between two molecules, dueto, for example, covalent, electrostatic, hydrophobic, and ionic and/orhydrogen-bond interactions under physiological conditions, and includesinteractions such as salt bridges and water bridges, as well as anyother conventional means of binding.

Antibodies may be screened for binding affinity by methods known in theart including, but not limited to, gel-shift assays, Western blots,radiolabeled competition assay, co-fractionation by chromatography,co-precipitation, cross linking, ELISA, and the like, which aredescribed in, for example, Current Protocols in Molecular Biology (1999)John Wiley & Sons, NY, which is incorporated herein by reference in itsentirety.

Antibodies which bind to the desired epitope on the target antigen maybe screened in a routine cross-blocking assay such as described inAntibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, EdHarlow and David Lane (1988), can be performed. Routine competitivebinding assays may also be used, in which an unknown antibody ischaracterized by its ability to inhibit binding of target to atarget-specific antibody of the invention. Intact antigen, fragmentsthereof such as the extracellular domain, or linear epitopes can beused. Epitope mapping is described in Champe et al., J. Biol. Chem. 270:1388-1394 (1995).

Antibodies that inhibit or neutralize human hepcidin activity may beidentified by contacting hepcidin with an antibody, comparing hepcidinactivity in the presence and absence of the test antibody, anddetermining whether the presence of the antibody decreases activity ofthe hepcidin. The biological activity of a particular antibody, orcombination of antibodies, may be evaluated in vivo using a suitableanimal model, including any of those described herein.

In one embodiment, provided herein are high throughput screening (HTS)assays to identify antibodies that interact with or inhibit biologicalactivity of target hepcidin. HTS assays permit screening of largenumbers of compounds in an efficient manner.

The phrase “conservative amino acid substitution” refers to grouping ofamino acids on the basis of certain common properties. A functional wayto define common properties between individual amino acids is to analyzethe normalized frequencies of amino acid changes between correspondingproteins of homologous organisms (Schulz, G. E. and R. H. Schirmer,Principles of Protein Structure, Springer-Verlag). According to suchanalyses, groups of amino acids may be defined where amino acids withina group exchange preferentially with each other, and therefore resembleeach other most in their impact on the overall protein structure(Schulz, G. E. and R. H. Schirmer, Principles of Protein Structure,Springer-Verlag). Examples of amino acid groups defined in this mannerinclude:

(i) a charged group, consisting of Glu and Asp, Lys, Arg and His,

(ii) a positively-charged group, consisting of Lys, Arg and His,

(iii) a negatively-charged group, consisting of Glu and Asp,

(iv) an aromatic group, consisting of Phe, Tyr and Trp,

(v) a nitrogen ring group, consisting of His and Trp,

(vi) a large aliphatic non-polar group, consisting of Val, Leu and Ile,

(vii) a slightly-polar group, consisting of Met and Cys,

(viii) a small-residue group, consisting of Ser, Thr, Asp, Asn, Gly,Ala, Glu, Gln and Pro,

(ix) an aliphatic group consisting of Val, Leu, Ile, Met and Cys, and

(x) a small hydroxyl group consisting of Ser and Thr.

In addition to the groups presented above, each amino acid residue mayform its own group, and the group formed by an individual amino acid maybe referred to simply by the one and/or three letter abbreviation forthat amino acid commonly used in the art as described above.

A “conserved residue” is an amino acid that is relatively invariantacross a range of similar proteins. Often conserved residues will varyonly by being replaced with a similar amino acid, as described above for“conservative amino acid substitution.”

The letter “x” or “xaa” as used in amino acid sequences herein isintended to indicate that any of the twenty standard amino acids may beplaced at this position unless specifically noted otherwise.

“Homology” or “identity” or “similarity” refers to sequence similaritybetween two peptides or between two nucleic acid molecules. Homology andidentity can each be determined by comparing a position in each sequencewhich may be aligned for purposes of comparison. When an equivalentposition in the compared sequences is occupied by the same base or aminoacid, then the molecules are identical at that position; when theequivalent site occupied by the same or a similar amino acid residue(e.g., similar in steric and/or electronic nature), then the moleculescan be referred to as homologous (similar) at that position. Expressionas a percentage of homology/similarity or identity refers to a functionof the number of identical or similar amino acids at positions shared bythe compared sequences. A sequence which is “unrelated” or“non-homologous” shares less than 40% identity, though preferably lessthan 25% identity with a sequence of the present invention. In comparingtwo sequences, the absence of residues (amino acids or nucleic acids) orpresence of extra residues also decreases the identity andhomology/similarity.

The term “homology” describes a mathematically based comparison ofsequence similarities which is used to identify genes or proteins withsimilar functions or motifs. The nucleic acid (nucleotide,oligonucleotide) and amino acid (protein) sequences of the presentinvention may be used as a “query sequence” to perform a search againstpublic databases to, for example, identify other family members, relatedsequences or homologs. Such searches can be performed using the NBLASTand XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol.Biol. 215:403-10. BLAST nucleotide searches can be performed with theNBLAST program, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to nucleic acid molecules of the invention. BLAST amino acidsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to proteinmolecules of the invention. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilized as described in Altschul et al.,(1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST andGapped BLAST programs, the default parameters of the respective programs(e.g., XBLAST and BLAST) can be used (see, www.ncbi.nlm.nih.gov).

As used herein, “identity” means the percentage of identical nucleotideor amino acid residues at corresponding positions in two or moresequences when the sequences are aligned to maximize sequence matching,i.e., taking into account gaps and insertions. Identity can be readilycalculated by known methods, including but not limited to thosedescribed in (Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073(1988). Methods to determine identity are designed to give the largestmatch between the sequences tested. Moreover, methods to determineidentity are codified in publicly available computer programs. Computerprogram methods to determine identity between two sequences include, butare not limited to, the GCG program package (Devereux, J., et al.,Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA(Altschul, S. F. et al., J. Molec. Biol. 215: 403-410 (1990) andAltschul et al. Nuc. Acids Res. 25: 3389-3402 (1997)). The BLAST Xprogram is publicly available from NCBI and other sources (BLAST Manual,Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., etal., J. Mol. Biol. 215: 403-410 (1990). The well known Smith Watermanalgorithm may also be used to determine identity.

“Isolated” (used interchangeably with “substantially pure” or“purified”) when applied to polypeptides means a polypeptide or aportion thereof which, by virtue of its origin or manipulation: (i) ispresent in a host cell as the expression product of a portion of anexpression vector; or (ii) is linked to a protein or other chemicalmoiety other than that to which it is linked in nature; or (iii) doesnot occur in nature, for example, a protein that is chemicallymanipulated by appending, or adding at least one hydrophobic moiety tothe protein so that the protein is in a form not found in nature. By“isolated” it is further meant a protein that is: (i) synthesizedchemically; or (ii) expressed in a host cell and purified away fromassociated and contaminating proteins. The term generally means apolypeptide that has been separated from other proteins and nucleicacids with which it naturally occurs. Preferably, the polypeptide isalso separated from substances such as antibodies or gel matrices(polyacrylamide) which are used to purify it.

“Inducing a host immune response” means that a subject experiencesalleviation or reduction of signs or symptoms of illness, andspecifically includes, without limitation, prolongation of survival.

Humanized immunoglobulins, including humanized antibodies, have beenconstructed by means of genetic engineering. Most humanizedimmunoglobulins that have been previously described have comprised aframework that is identical to the framework of a particular humanimmunoglobulin chain (i.e., an acceptor or recipient), and three CDRsfrom a non-human (i.e., donor) immunoglobulin chain. As describedherein, humanization can also include criteria by which a limited numberof amino acids in the framework of a humanized immunoglobulin chain areidentified and chosen to be the same as the amino acids at thosepositions in the donor rather than in the acceptor, in order to increasethe affinity of an antibody comprising the humanized immunoglobulinchain.

When increased affinity of a humanized antibody is desired, residueswithin the CDRs of a converted antibody may be additionally substitutedwith other amino acids. Typically, no more than four amino acid residuesin a CDR are changed, and most typically no more than two residues inthe CDR will be changed, except for heavy chain CDR2, where as many as10 residues may be changed. Changes in affinity can be measured byconventional methods such as those described herein (e.g., Biacore).

Methods of “superhumanizing” antibodies are described in more detail inU.S. Pat. No. 6,881,557 which is hereby incorporated by reference in itsentirety.

Humanized antibodies and antigen-binding fragments can be constructedand produced using conventional techniques known in the art. Inaddition, recombinantly prepared antibodies can often be produced inlarge quantities, particularly when utilizing high level expressionvectors.

Antibodies can be sequenced using conventional techniques known in theart. In one aspect, the amino acid sequences of one or more of the CDRsis inserted into a synthetic sequence of, for example, a human antibody(or antigen-binding fragment thereof) framework to create a humanantibody that could limit adverse side reactions of treating a humansubject with a non-human antibody. The amino acid sequences of one ormore of the CDRs can also be inserted into a synthetic sequence of, forexample, into a binding protein such as an AVIMER™ to create a constructfor administration to a human subject. Such techniques can be modifieddepending on the species of animal to be treated. For example, forveterinary uses, an antibody, antigen-binding fragment or bindingprotein can be synthesized for administration of a non-human (e.g., aprimate, a cow, a horse, etc.).

In another aspect, using art-recognized techniques such as thoseprovided and incorporated herein, nucleotides encoding amino acidsequences of one or more of the CDRs can inserted, for example, byrecombinant techniques in restriction endonuclease sites of an existingpolynucleotide that encodes an antibody, antigen-binding fragment orbinding protein.

For expression, an expression system is one which utilizes the GS system(Lonza) using a glutamine synthetase gene as the selectable marker.Briefly, a transfection is performed in CHO cells by electroporation(250V) using the GS system (Lonza) using the glutamine synthetase geneas the selectable marker. Wild type CHO cells are grown in DMEM (Sigma)containing 10% dialyzed Fetal Calf Serum (FCS) with 2 mM glutamine.6×10⁷ CHO cells are transfected with 300 μg of linearized DNA byelectroporation. After electroporation the cells are resuspended in DMEMwith glutamine and plated out into 36×96-well plates (50 μl/well), andincubated at 37° C. in 5% CO₂. The following day, 150 μl/well ofselective medium (DMEM without glutamine) is added. After approximately3 weeks the colonies are screened by ELISA (see below) using anirrelevant antibody as a negative control. All colonies producing >20μg/ml are expanded into 24-well plates and then into duplicate T25flasks.

For high level production, the most widely used mammalian expressionsystem is one which utilizes the gene amplification procedure offered bydehydrofolate reductase deficient (“dhfr-”) Chinese hamster ovary cells.The system is well known to the skilled artisan. The system is basedupon the dehydrofolate reductase “dhfr” gene, which encodes the DHFRenzyme, which catalyzes conversion of dehydrofolate to tetrahydrofolate.In order to achieve high production, dhfr-CHO cells are transfected withan expression vector containing a functional DHFR gene, together with agene that encodes a desired protein. In this case, the desired proteinis recombinant antibody heavy chain and/or light chain.

By increasing the amount of the competitive DHFR inhibitor methotrexate(MTX), the recombinant cells develop resistance by amplifying the dhfrgene. In standard cases, the amplification unit employed is much largerthan the size of the dhfr gene, and as a result the antibody heavy chainis co-amplified.

When large scale production of the protein, such as the antibody chain,is desired, both the expression level and the stability of the cellsbeing employed are taken into account. In long term culture, recombinantCHO cell populations lose homogeneity with respect to their specificantibody productivity during amplification, even though they derive froma single, parental clone.

The present application provides an isolated polynucleotide (nucleicacid) encoding an antibody or antigen-binding fragment as describedherein, vectors containing such polynucleotides, and host cells andexpression systems for transcribing and translating such polynucleotidesinto polypeptides.

The present application also provides constructs in the form ofplasmids, vectors, transcription or expression cassettes which compriseat least one polynucleotide as above.

The present application also provides a recombinant host cell whichcomprises one or more constructs as above. A nucleic acid encoding anyantibody or antigen-binding fragments thereof described herein asprovided itself forms an aspect of the present application, as does amethod of production of the antibody or antigen-binding fragmentsthereof described herein which method comprises expression from encodingnucleic acid therefrom. Expression can conveniently be achieved byculturing under appropriate conditions recombinant host cells containingthe nucleic acid. Following production by expression, an antibody orantigen-binding fragment can be isolated and/or purified using anysuitable technique, then used as appropriate.

Specific antibodies, antigen-binding fragments, and encoding nucleicacid molecules and vectors described herein can be provided isolatedand/or purified, e.g., from their natural environment, in substantiallypure or homogeneous form. In the case of nucleic acid, free orsubstantially free of nucleic acid or genes origin other than thesequence encoding a polypeptide with the required function. Nucleic acidcan comprise DNA or RNA and can be wholly or partially synthetic.Methods of purification are well known in the art.

Systems for cloning and expression of a polypeptide in a variety ofdifferent host cells are well known. Suitable host cells include, butare not limited to, bacteria cells, mammalian cells, yeast cells andbaculovirus systems. Mammalian cell lines available in the art forexpression of a heterologous polypeptide include Chinese hamster ovarycells, HeLa cells, baby hamster kidney cells, NS0 mouse myeloma cellsand many others. A common bacterial host is E. coli.

The expression of antibodies and antibody fragments in prokaryotic cellssuch as E. coli is well established in the art. For a review, see forexample Plückthun, A. Bio/Technology 9: 545-551 (1991). Expression ineukaryotic cells in culture is also available to those skilled in theart as an option for production of the antibodies and antigen-bindingfragments described herein, see for recent reviews, for example Raff, M.E. (1993) Curr. Opinion Biotech. 4: 573-576; Trill J. J. et al. (1995)Curr. Opinion Biotech 6: 553-560, each of which is which is incorporatedherein by reference in its entirety.

Suitable vectors can be chosen or constructed, containing appropriateregulatory sequences, including promoter sequences, terminatorsequences, polyadenylation sequences, enhancer sequences, marker genesand other sequences as appropriate. Vectors can be plasmids, viral e.g.‘phage, or phagemid, as appropriate. For further details see, forexample, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrooket al., 1989, Cold Spring Harbor Laboratory Press. Many known techniquesand protocols for manipulation of nucleic acid, for example inpreparation of nucleic acid constructs, mutagenesis, sequencing,introduction of DNA into cells and gene expression, and analysis ofproteins, are described in detail in Short Protocols in MolecularBiology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992.The methods disclosures of Sambrook et al. and Ausubel et al. areincorporated herein by reference in their entirety and are well known inthe art.

Thus, a further aspect provides a host cell containing nucleic acid asdisclosed herein. A still further aspect provides a method comprisingintroducing such nucleic acid into a host cell. The introduction canemploy any available technique. For eukaryotic cells, suitabletechniques can include, for example, calcium phosphate transfection,DEAE Dextran, electroporation, liposome-mediated transfection andtransduction using retrovirus or other virus, e.g., vaccinia or, forinsect cells, baculovirus. For bacterial cells, suitable techniques caninclude, for example, calcium chloride transformation, electroporationand transfection using bacteriophage.

The introduction can be followed by causing or allowing expression fromthe nucleic acid, e.g. by culturing host cells under conditions forexpression of the gene.

In one embodiment, the nucleic acid is integrated into the genome (e.g.chromosome) of the host cell. Integration can be promoted by inclusionof sequences which promote recombination with the genome, in accordancewith standard techniques. Ig enhances can be initialized as needed tomaximize expression.

The present application also provides a method which comprises using aconstruct as stated above in an expression system in order to expressthe antibodies or antigen-binding fragments thereof as above.

The present application also relates to isolated nucleic acids, such asrecombinant DNA molecules or cloned genes, or degenerate variantsthereof, mutants, analogs, or fragments thereof, which encode anantibody or antigen-binding sequence described herein.

In one aspect, the present application provides a nucleic acid whichcodes for an antibody or antigen-binding fragment thereof as describedherein.

In a further embodiment, the full DNA sequence of the recombinant DNAmolecule or cloned gene of an antibody or antigen-binding fragmentdescribed herein can be operatively linked to an expression controlsequence which can be introduced into an appropriate host. Theapplication accordingly extends to unicellular hosts transformed withthe cloned gene or recombinant DNA molecule comprising a DNA sequenceencoding the V_(H) and/or V_(L), or portions thereof, of the antibody.

Another feature is the expression of the DNA sequences disclosed herein.As is well known in the art, DNA sequences can be expressed byoperatively linking them to an expression control sequence in anappropriate expression vector and employing that expression vector totransform an appropriate unicellular host.

Such operative linking of a DNA sequence to an expression controlsequence, of course, includes, if not already part of the DNA sequence,the provision of an initiation codon, ATG, in the correct reading frameupstream of the DNA sequence.

Polynucleotides and vectors can be provided in an isolated and/or apurified form (e.g., free or substantially free of polynucleotides oforigin other than the polynucleotide encoding a polypeptide with therequired function). As used herein, “substantially pure,” and“substantially free” refer to a solution or suspension containing lessthan, for example, about 20% or less extraneous material, about 10% orless extraneous material, about 5% or less extraneous material, about 4%or less extraneous material, about 3% or less extraneous material, about2% or less extraneous material, or about 1% or less extraneous material.

A wide variety of host/expression vector combinations can be employed inexpressing the DNA sequences of this invention. Useful expressionvectors, for example, can consist of segments of chromosomal,non-chromosomal and synthetic DNA sequences. Suitable vectors include,but are not limited to, derivatives of SV40 and known bacterialplasmids, e.g., E. coli plasmids col E1, Pcr1, Pbr322, Pmb9 and theirderivatives, plasmids such as RP4; phage DNAs, e.g., the numerousderivatives of phage λ, e.g., NM989, and other phage DNA, e.g., M13 andfilamentous single stranded phage DNA; yeast plasmids such as the 2uplasmid or derivatives thereof; vectors useful in eukaryotic cells, suchas vectors useful in insect or mammalian cells; vectors derived fromcombinations of plasmids and phage DNAs, such as plasmids that have beenmodified to employ phage DNA or other expression control sequences; andthe like.

Also provided herein is a recombinant host cell which comprises one ormore polynucleotide constructs. A polynucleotide encoding an antibody orantigen-binding fragment as provided herein forms an aspect of thepresent application, as does a method of production of the antibody orantigen-binding fragment which method comprises expression from thepolynucleotide. Expression can be achieved, for example, by culturingunder appropriate conditions recombinant host cells containing thepolynucleotide. An antibody or antigen-binding fragment can then beisolated and/or purified using any suitable technique, and used asappropriate.

Any of a wide variety of expression control sequences—sequences thatcontrol the expression of a DNA sequence operatively linked to it—can beused in these vectors to express the DNA sequences. Such usefulexpression control sequences include, for example, the early or latepromoters of SV40, CMV, vaccinia, polyoma or adenovirus, the lac system,the trp system, the TAC system, the TRC system, the LTR system, themajor operator and promoter regions of phage λ, the control regions offd coat protein, the promoter for 3-phosphoglycerate kinase or otherglycolytic enzymes, the promoters of acid phosphatase (e.g., Pho5), thepromoters of the yeast alpha-mating factors, and other sequences knownto control the expression of genes of prokaryotic or eukaryotic cells ortheir viruses, and various combinations thereof.

Systems for cloning and expression of a polypeptide in a variety ofdifferent host cells are well known. Suitable host cells includebacteria, mammalian cells, yeast and baculovirus systems. Mammalian celllines available in the art for expression of a heterologous polypeptideinclude Chinese hamster ovary (CHO) cells, HeLa cells, baby hamsterkidney cells, NS0 mouse myeloma cells and many others. A common,bacterial host can be, for example, E. coli.

The expression of antibodies or antigen-binding fragments in prokaryoticcells, such as E. coli, is well established in the art. For a review,see for example Plückthun, A. Bio/Technology 9: 545-551 (1991).Expression in eukaryotic cells in culture is also available to thoseskilled in the art (Raff, M. E. (1993) Curr. Opinion Biotech. 4:573-576; Trill J. J. et al. (1995) Curr. Opinion Biotech 6: 553-560).

A wide variety of unicellular host cells are also useful in expressingthe DNA sequences. These hosts include well-known eukaryotic andprokaryotic hosts, such as strains of E. coli, Pseudomonas, Bacillus,Streptomyces, fungi such as yeasts, and animal cells, such as CHO,YB/20, NS0, SP2/0, R1.1, B-W and L-M cells, African Green Monkey kidneycells (e.g., COS 1, COS 7, BSC1, BSC40, and BMT10), insect cells (e.g.,Sf9), and human cells and plant cells in tissue culture.

It will be understood that not all vectors, expression control sequencesand hosts will function equally well to express the DNA sequences.Neither will all hosts function equally well with the same expressionsystem. However, one skilled in the art will be able to select theproper vectors, expression control sequences, and hosts without undueexperimentation to accomplish the desired expression without departingfrom the scope of this application. For example, in selecting a vector,the host must be considered because the vector must function in it. Thevector's copy number, the ability to control that copy number, and theexpression of any other proteins encoded by the vector, such asantibiotic markers, will also be considered. One of ordinary skill inthe art can select the proper vectors, expression control sequences, andhosts to accomplish the desired expression without departing from thescope of this application. For example, in selecting a vector, the hostis considered because the vector functions in it. The vector's copynumber, the ability to control that copy number, and the expression ofany other proteins encoded by the vector, such as antibiotic markers,can also be considered.

The present application also provides constructs in the form ofplasmids, vectors, transcription or expression cassettes as describedelsewhere herein which comprise at least one polynucleotide as above.Suitable vectors can be chosen or constructed, containing appropriateregulatory sequences, including promoter sequences, terminatorsequences, polyadenylation sequences, enhancer sequences, selectablemarkers and other sequences as appropriate. Vectors can be plasmids,viral e.g., phage, phagemid, etc., as appropriate. For further detailssee, for example, Molecular Cloning: a Laboratory Manual: 2nd edition,Sambrook et al., 1989, Cold Spring Harbor Laboratory Press. Many knowntechniques and protocols for manipulation of nucleic acid, for examplein preparation of nucleic acid constructs, mutagenesis, sequencing,introduction of DNA into cells and gene expression, and analysis ofproteins, are described in detail in Short Protocols in MolecularBiology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992.The methods and disclosures of Sambrook et al. and Ausubel et al. areincorporated herein by reference.

In selecting an expression control sequence, a variety of factors willnormally be considered. These include, for example, the relativestrength of the system, its controllability, and its compatibility withthe particular DNA sequence or gene to be expressed, particularly asregards potential secondary structures. Suitable unicellular hosts willbe selected by consideration of, e.g., their compatibility with thechosen vector, their secretion characteristics, their ability to foldproteins correctly, and their fermentation requirements, as well as thetoxicity to the host of the product encoded by the DNA sequences to beexpressed, and the ease of purification of the expression products.

A further aspect provides a host cell containing one or morepolynucleotides as disclosed herein. Yet a further aspect provides amethod of introducing such one or more polynucleotides into a host cell,any available technique. For eukaryotic cells, suitable techniques caninclude, for example, calcium phosphate transfection, DEAEDextran,electroporation, liposome-mediated transfection and transduction usingretrovirus or other virus (e.g. vaccinia) or, for insect cells,baculovirus. For bacterial cells, suitable techniques can include, forexample calcium chloride transformation, electroporation andtransfection using bacteriophages.

The introduction can be followed by causing or allowing expression fromthe one or more polynucleotides, e.g. by culturing host cells underconditions for expression of one or more polypeptides from one or morepolynucleotides. Inducible systems can be used and expression induced byaddition of an activator.

In one embodiment, the polynucleotides can be integrated into the genome(e.g., chromosome) of the host cell. Integration can be promoted byinclusion of sequences which promote recombination with the genome, inaccordance with standard techniques. In another embodiment, the nucleicacid is maintained on an episomal vector in the host cell.

Methods are provided herein which include using a construct as statedabove in an expression system in order to express a specificpolypeptide.

Considering these and other factors, a person skilled in the art will beable to construct a variety of vector/expression control sequence/hostcombinations that will express the DNA sequences on fermentation or inlarge scale animal culture.

A polynucleotide encoding an antibody, antigen-binding fragment, or abinding protein can be prepared recombinantly/synthetically in additionto, or rather than, cloned. The polynucleotide can be designed with theappropriate codons for the antibody, antigen-binding fragment, or abinding protein. In general, one will select preferred codons for anintended host if the sequence will be used for expression. The completepolynucleotide can be assembled from overlapping oligonucleotidesprepared by standard methods and assembled into a complete codingsequence. See, e.g., Edge, Nature, 292:756 (1981); Nambair et al.,Science, 223:1299 (1984); Jay et al., J. Biol. Chem., 259:6311 (1984),each of which is which is incorporated herein by reference in itsentirety.

A general method for site-specific incorporation of unnatural aminoacids into proteins is described in Noren et al., Science, 244:182-188(April 1989). This method can be used to create analogs with unnaturalamino acids.

As mentioned above, a DNA sequence encoding an antibody orantigen-binding fragment thereof can be prepared synthetically ratherthan cloned. The DNA sequence can be designed with the appropriatecodons for the antibody or antigen-binding fragment amino acid sequence.In general, one will select preferred codons for the intended host ifthe sequence will be used for expression. The complete sequence isassembled from overlapping oligonucleotides prepared by standard methodsand assembled into a complete coding sequence.

Antibodies, or antigen-binding fragments thereof, can be modified usingtechniques known in the art for various purposes such as, for example,by addition of polyethylene glycol (PEG). PEG modification (PEGylation)can lead to one or more of improved circulation time, improvedsolubility, improved resistance to proteolysis, reduced antigenicity andimmunogenicity, improved bioavailability, reduced toxicity, improvedstability, and easier formulation (for a review see, Francis et al.,International Journal of Hematology 68:1-18, 1998).

In the case of an antigen-binding fragment which does not contain an Fcportion, an Fc portion can be added to (e.g., recombinantly) thefragment, for example, to increase half-life of the antigen-bindingfragment in circulation in blood when administered to a subject. Choiceof an appropriate Fc region and methods of to incorporate such fragmentsare known in the art. Incorporating a Fc region of an IgG into apolypeptide of interest so as to increase its circulatory half-life, butso as not to lose its biological activity can be accomplished usingconventional techniques known in the art such as, for example, describedin U.S. Pat. No. 6,096,871, which is hereby incorporated by reference inits entirety. Fc portions of antibodies can be further modified toincrease half-life of the antigen-binding fragment in circulation inblood when administered to a subject. Modifications can be determinedusing conventional means in the art such as, for example, described inU.S. Pat. No. 7,217,798, which is hereby incorporated by reference inits entirety.

Other methods of improving the half-life of antibody-based fusionproteins in circulation are also known such as, for example, describedin U.S. Pat. Nos. 7,091,321 and 6,737,056, each of which is herebyincorporated by reference. Additionally, antibodies and antigen-bindingfragments thereof may be produced or expressed so that they do notcontain fucose on their complex N-glycoside-linked sugar chains. Theremoval of the fucose from the complex N-glycoside-linked sugar chainsis known to increase effector functions of the antibodies andantigen-binding fragments, including but not limited to, antibodydependent cell-mediated cytotoxicity (ADCC) and complement dependentcytotoxicity (CDC). Similarly, antibodies or antigen-binding fragmentsthereof that can bind an epitope can be attached at their C-terminal endto all or part of an immunoglobulin heavy chain derived from anyantibody isotype, e.g., IgG, IgA, IgE, IgD and IgM and any of theisotype sub-classes, particularly IgG1, IgG2b, IgG2a, IgG3 and IgG4.

Additionally, the antibodies or antigen-binding fragments describedherein can also be modified so that they are able to cross theblood-brain barrier. Such modification of the antibodies orantigen-binding fragments described herein allows for the treatment ofbrain diseases such as glioblastoma multiforme (GBM). Exemplarymodifications to allow proteins such as antibodies or antigen-bindingfragments to cross the blood-brain barrier are described in US PatentApplication Publication 2007/0082380 which is hereby incorporated byreference in its entirety.

Glycosylation of immunoglobulins has been shown to have significanteffects on their effector functions, structural stability, and rate ofsecretion from antibody-producing cells (Leatherbarrow et al., Mol.Immunol. 22:407 (1985)). The carbohydrate groups responsible for theseproperties are generally attached to the constant (C) regions of theantibodies. For example, glycosylation of IgG at asparagine 297 in theC_(H)2 domain is required for full capacity of IgG to activate theclassical pathway of complement-dependent cytolysis (Tao and Morrison,J. Immunol. 143:2595 (1989)). Glycosylation of IgM at asparagine 402 inthe C_(H)3 domain is necessary for proper assembly and cytolyticactivity of the antibody (Muraoka and Shulman, J. Immunol. 142:695(1989)). Removal of glycosylation sites as positions 162 and 419 in theC_(H)1 and C_(H)3 domains of an IgA antibody led to intracellulardegradation and at least 90% inhibition of secretion (Taylor and Wall,Mol. Cell. Biol. 8:4197 (1988)). Additionally, antibodies andantigen-binding fragments thereof may be produced or expressed so thatthey do not contain fucose on their complex N-glycoside-linked sugarchains. The removal of the fucose from the complex N-glycoside-linkedsugar chains is known to increase effector functions of the antibodiesand antigen-binding fragments, including but not limited to, antibodydependent cell-mediated cytotoxicity (ADCC) and complement dependentcytotoxicity (CDC). These “defucosylated” antibodies and antigen-bindingfragments may be produced through a variety of systems utilizingmolecular cloning techniques known in the art, including but not limitedto, transgenic animals, transgenic plants, or cell-lines that have beengenetically engineered so that they no longer contain the enzymes andbiochemical pathways necessary for the inclusion of a fucose in thecomplex N-glycoside-linked sugar chains (also known asfucosyltransferase knock-out animals, plants, or cells). Non-limitingexamples of cells that can be engineered to be fucosyltransferaseknock-out cells include CHO cells, SP2/0 cells, NS0 cells, and YB2/0cells.

Glycosylation of immunoglobulins in the variable (V) region has alsobeen observed. Sox and Hood reported that about 20% of human antibodiesare glycosylated in the V region (Proc. Natl. Acad. Sci. USA 66:975(1970)). Glycosylation of the V domain is believed to arise fromfortuitous occurrences of the N-linked glycosylation signalAsn-Xaa-Ser/Thr in the V region sequence and has not been recognized inthe art as playing a role in immunoglobulin function.

Glycosylation at a variable domain framework residue can alter thebinding interaction of the antibody with antigen. The present inventionincludes criteria by which a limited number of amino acids in theframework or CDRs of a humanized immunoglobulin chain are chosen to bemutated (e.g., by substitution, deletion, or addition of residues) inorder to increase the affinity of an antibody.

Cysteine residue(s) may be removed or introduced in the Fc region of anantibody or Fc-containing polypeptide, thereby eliminating or increasinginterchain disulfide bond formation in this region. A homodimericspecific binding agent or antibody generated using such methods mayexhibit improved internalization capability and/or increasedcomplement-mediated cell killing and antibody-dependent cellularcytotoxicity (ADCC). See Caron et al., J. Exp Med. 176: 1191-1195 (1992)and Shopes, B. J. Immunol. 148: 2918-2922 (1992).

It has been shown that sequences within the CDR can cause an antibody tobind to MHC Class II and trigger an unwanted helper T-cell response. Aconservative substitution may allow the antibody to retain bindingactivity, yet reduce its ability to trigger an unwanted T-cell response.In one embodiment, one or more of the N-terminal 20 amino acids of theheavy or light chain may be removed.

In some embodiments, antibody molecules may be produced with alteredcarbohydrate structure resulting in altered effector activity, includingantibody molecules with absent or reduced fucosylation that exhibitimproved ADCC activity. A variety of ways are known in the art toaccomplish this. For example, ADCC effector activity is mediated bybinding of the antibody molecule to the FcγRIII receptor, which has beenshown to be dependent on the carbohydrate structure of the N-linkedglycosylation at the Asn-297 of the CH₂ domain. Non-fucosylatedantibodies bind this receptor with increased affinity and triggerFcγRIII-mediated effector functions more efficiently than native,fucosylated antibodies. Some host cell strains, e.g. Lec13 or rathybridoma YB2/0 cell line naturally produce antibodies with lowerfucosylation levels. Shields et al., J Biol Chem. Jul. 26, 2002;277(30):26733-40; Shinkawa et al., J Biol Chem. Jan. 31, 2003;278(5):3466-73. An increase in the level of bisected carbohydrate, e.g.through recombinantly producing antibody in cells that overexpressGnTIII enzyme, has also been determined to increase ADCC activity. Umanaet al., Nat Biotechnol. February 1999; 17(2):176-80. It has beenpredicted that the absence of only one of the two fucose residues may besufficient to increase ADCC activity. (Ferrara et al., J Biol Chem. Dec.5, 2005).

Covalent modifications of an antibody are also included herein. They maybe made by chemical synthesis or by enzymatic or chemical cleavage ofthe antibody, if applicable. Other types of covalent modifications maybe introduced by reacting targeted amino acid residues with an organicderivatizing agent that is capable of reacting with selected side chainsor the N- or C-terminal residues.

Cysteinyl residues most commonly are reacted with alpha-haloacetates(and corresponding amines), such as chloroacetic acid orchloroacetamide, to give carboxymethyl or carboxyamidomethylderivatives. Cysteinyl residues also are derivatized by reaction withbromotrifluoroacetone, alpha-bromo-beta-(5-imidozoyl)propionic acid,chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide,methyl 2-pyridyl disulfide, p-chloromercuribenzoate,2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues may be derivatized by reaction withdiethylpyrocarbonate at pH 5.5-7.0 because this agent is relativelyspecific for the histidyl side chain. Para-bromophenacyl bromide also isuseful; the reaction may be performed in 0.1 M sodium cacodylate at pH6.0.

Lysinyl and amino-terminal residues may be reacted with succinic orother carboxylic acid anhydrides. Derivatization with these agents hasthe effect of reversing the charge of the lysinyl residues. Othersuitable reagents for derivatizing alpha-amino-containing residuesinclude imidoesters such as methyl picolinimidate, pyridoxal phosphate,pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid,O-methylisourea, 2,4-pentanedione, and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues may be modified by reaction with one or severalconventional reagents, such as phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pK_(a) of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues may be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidizole and tetranitromethane may be used to formO-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosylresidues are iodinated using ¹²⁵I or 131I to prepare labeled proteinsfor use in radioimmunoassay.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R—N═C═N—R′), where R and R′ are differentalkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide.Furthermore, aspartyl and glutamyl residues are converted to asparaginyland glutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues may be deamidated to thecorresponding glutamyl and aspartyl residues, respectively. Theseresidues are deamidated under neutral or basic conditions.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the alpha-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MolecularProperties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)),acetylation of the N-terminal amine, and amidation of any C-terminalcarboxyl group.

Another type of covalent modification involves chemically orenzymatically coupling glycosides to the specific binding agent orantibody. These procedures are advantageous in that they do not requireproduction of the polypeptide or antibody in a host cell that hasglycosylation capabilities for N- or O-linked glycosylation. Dependingon the coupling mode used, the sugar(s) may be attached to (a) arginineand histidine, (b) free carboxyl groups, (c) free sulfhydryl groups suchas those of cysteine, (d) free hydroxyl groups such as those of serine,threonine, or hydroxyproline, (e) aromatic residues such as those ofphenylalanine, tyrosine, or tryptophan, or (f) the amide group ofglutamine. These methods are described in WO 87/05330 published 11 Sep.1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306(1981).

Removal of any carbohydrate moieties present on the polypeptide orantibody may be accomplished chemically or enzymatically. Chemicaldeglycosylation involves exposure of the antibody to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving theantibody intact. Chemical deglycosylation is described by Hakimuddin, etal. Arch. Biochem. Biophys. 259: 52 (1987) and by Edge et al. Anal.Biochem., 118: 131 (1981). Enzymatic cleavage of carbohydrate moietieson an antibody can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al. Meth. Enzymol. 138:350 (1987).

Another type of covalent modification of hepcidin activity compriseslinking an antibody to one of a variety of nonproteinaceous polymers,e.g., polyethylene glycol, polypropylene glycol, polyoxyethylatedpolyols, polyoxyethylated sorbitol, polyoxyethylated glucose,polyoxyethylated glycerol, polyoxyalkylenes, or polysaccharide polymerssuch as dextran. Such methods are known in the art, see, e.g. U.S. Pat.Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192, 4,179,337,4,766,106, 4,179,337, 4,495,285, 4,609,546 or EP 315 456.

Affinity for binding a pre-determined polypeptide antigen can,generally, be modulated by introducing one or more mutations into the Vregion framework, typically in areas adjacent to one or more CDRs and/orin one or more framework regions. Typically, such mutations involve theintroduction of conservative amino acid substitutions that eitherdestroy or create the glycosylation site sequences but do notsubstantially affect the hydropathic structural properties of thepolypeptide. Typically, mutations that introduce a proline residue areavoided. Glycosylation of antibodies and antigen-binding fragmentsthereof is further described in U.S. Pat. No. 6,350,861, which isincorporated by reference herein with respect to glycosylation.

Anti-Hepcidin Antibodies

Provided herein are humanized antibodies, and antigen-binding fragmentsthereof that bind hepcidin.

Hepcidin is involved in regulating iron homeostasis. Hepcidin binds toferroportin and decreases its functional activity by causing it to beinternalized from the cell surface and degraded.

High levels of human hepcidin may result in reduced iron levels, andvice versa. Mutations in the hepcidin gene which result in lack ofhepcidin activity are associated with juvenile hemochromatosis, a severeiron overload disease. Studies in mice have demonstrated a role ofhepcidin in control of normal iron homeostasis.

Hepcidin may also be involved in iron sequestration during inflammation.Hepcidin gene expression has been observed to be robustly up-regulatedafter inflammatory stimuli, such as infections, which induce the acutephase response of the innate immune systems of vertebrates. Hepcidingene expression may be up-regulated by lipopolysaccharide (LPS),turpentine, Freund's complete adjuvant, incomplete adjuvant, adenoviralinfections and the inflammatory cytokine interleukin-6 (IL-6). A strongcorrelation between hepcidin expression and anemia of inflammation wasalso found in patients with chronic inflammatory diseases, includingbacterial, fungal, and viral infections.

Human hepcidin is a 25 amino acid peptide with anti-microbial andiron-regulating activity. It has also been referred to as LEAP-1(liver-expressed antimicrobial peptide). A hepcidin cDNA encoding an 83amino acid pre-propeptide in mice and an 84 amino acid pre-propeptide inrat and human were subsequently identified in a search for liverspecific genes that were regulated by iron. The 24 residue N-terminalsignal peptide is first cleaved to produce pro-hepcidin, which is thenfurther processed to produce mature hepcidin, found in both blood andurine. In human urine, the predominant form contains 25 amino acids,although shorter 22 and 20 amino acid peptides are also present atundetectable or very low concentrations in certain diseases.

Monoclonal antibodies (MAbs) have been raised against hepcidin whichmodulate hepcidin activity and thereby regulate iron homeostasis.Hereinafter, a reference to the terms “antibody” and “antibodies” are tobe considered inclusive of any of the antigen-binding fragmentsdescribed herein and the terms are to be interchangeable whereapplicable.

These antibodies, and antigen-binding fragments thereof, are useful forthe diagnosis and treatment of various conditions and diseases as wellas for purification and detection of hepcidin.

Binding of an antibody or antigen-binding fragment to hepcidin canpartially (e.g., 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,98%, 99% or any number therein) or completely modulate hepcidin. Theactivity of an antibody or antigen-binding fragment can be determinedusing an in vitro assay and/or in vivo using art-recognized assays suchas those described herein or otherwise known in the art.

In one aspect, the antigen-binding fragment of any one of the antibodiesdescribed above is a Fab, a Fab′, a Fd, a F(ab′)₂, a Fv, a scFv, asingle chain binding polypeptide (e.g., a scFv with Fc portion) or anyother functional fragment thereof as described herein.

Antibodies, or antigen-binding fragments thereof, described herein canbe further modified to alter the specific properties of the antibodywhile retaining the desired functionality, if needed. For example, inone embodiment, the compound can be modified to alter a pharmacokineticproperty of the compound, such as in vivo stability, solubility,bioavailability or half-life.

Antibodies, or antigen-binding fragments thereof, can be formulated forany suitable route of administration to a subject including, but notlimited to injection. Injection includes, for example, subcutaneous,peritoneal, or intravenous injection. Administration may be in one, two,three, four, five, six, seven, or more injection sites. In oneembodiment, administration is via six injection sites.

Antibodies, antigen-binding fragments, and binding proteins which bindhepcidin generated using such methods can be tested for one or more oftheir binding affinity, avidity, and modulating capabilities. Usefulantibodies, and antigen-binding fragments, can be administered to asubject to prevent, inhibit, manage or treat a condition disease ordisorder as described in more detail below.

Conventional methods may be utilized to identify antibodies orantigen-binding fragments thereof that bind to hepcidin. Antibodies andantigen-binding fragments can be evaluated for one or more of bindingaffinity, association rates, disassociation rates and avidity. In oneaspect, antibodies can be evaluated for their ability to modulate theactivity of hepcidin or a polypeptide in which the hepcidin bindingsequence (epitope) is present. Measurement binding affinity, associationrates, disassociation rates and avidity can be accomplished usingart-recognized assays including (Surface Plasmon Resonance), but notlimited to, an enzyme-linked-immunosorbent assay (ELISA), ScatchardAnalysis, BIACORE analysis, etc., as well as other assays commonly usedand known to those of ordinary skill in the art.

Measurement of binding of antibodies to hepcidin and/or the ability ofthe antibodies and antigen-binding fragments thereof, may be determinedusing, for example, an enzyme-linked-immunosorbent assay (ELISA), acompetitive binding assay, an ELISPOT assay, or any other useful assayknown in the art. These assays are commonly used and well-known to thoseof ordinary skill in the art.

In one non-limiting embodiment, an ELISA assay can be used to measurethe binding capability of specific antibodies or antigen-bindingfragments that bind to hepcidin.

Assays, such as an ELISA, also can be used to identify antibodies orantigen-binding fragments thereof which exhibit increased specificityfor hepcidin in comparison to other antibodies or antigen-bindingfragments thereof. Assays, such as an ELISA, also can be used toidentify antibodies or antigen-binding fragments thereof with bind toepitopes across one or more polypeptides and across one or more speciesof hepcidin. The specificity assay can be conducted by running parallelELISAs in which a test antibodies or antigen-binding fragments thereofis screened concurrently in separate assay chambers for the ability tobind one or more epitopes on different species of the polypeptidecontaining the hepcidin epitopes to identify antibodies orantigen-binding fragments thereof that bind to hepcidin. Anothertechnique for measuring apparent binding affinity familiar to those ofskill in the art is a surface plasmon resonance technique (analyzed on aBIACORE 2000 system) (Liljeblad, et al., Glyco. J. 2000, 17:323-329).Standard measurements and traditional binding assays are described byHeeley, R. P., Endocr. Res. 2002, 28:217-229.

Antibodies and antigen binding fragments thereof can be tested for avariety of functions using a variety of in vitro and in vivo methodsincluding, but not limited to those known in the art and those describedherein.

Provided herein are antibodies and antigen-binding fragments thereofthat bind to hepcidin. In one aspect, provided herein is an antibody, orantigen-binding fragment thereof, that specifically binds to hepcidin,comprising a heavy chain variable region and a light chain variableregion,

wherein said heavy chain variable region comprises:

(i) a CDR1 having an amino acid sequence of any one of SEQ ID NOS:55-57,

(ii) a CDR2 having an amino acid sequence of any one of SEQ ID NOS:58-60, and

(iii) a CDR3 having an amino acid sequence of any one of SEQ ID NOS:61-63;

and said light chain variable region comprises:

(i) a CDR1 having an amino acid sequence of any one of SEQ ID NOS:64-66,

(ii) a CDR2 having an amino acid sequence of any one of SEQ ID NOS:67-69, and

(iii) a CDR3 having an amino acid sequence of any one of SEQ ID NOS:70-72.

In one aspect, provided herein is an antibody, or antigen-bindingfragment thereof, that specifically binds to hepcidin or a hepcidinpeptide, comprising a heavy chain variable region and a light chainvariable region,

wherein said heavy chain variable region comprises:

(i) a CDR1 having an amino acid sequence encoded by any one of SEQ IDNOS: 1-3,

(ii) a CDR2 having an amino acid sequence encoded by any one of SEQ IDNOS: 4-6, and

(iii) a CDR3 having an amino acid sequence encoded by any one of SEQ IDNOS: 7-9;

and said light chain variable region comprises:

(i) a CDR1 having an amino acid sequence encoded by any one of SEQ IDNOS: 10-12,

(ii) a CDR2 having an amino acid sequence encoded by any one of SEQ IDNOS: 13-15, and

(iii) a CDR3 having an amino acid sequence encoded by any one of SEQ IDNOS: 16-18. In one aspect, provided herein is an antibody, orantigen-binding fragment thereof, comprises a heavy chain variableregion framework region; and a light chain variable region frameworkregion as set forth in the Sequence Listing below where the CDRsidentified in any one of SEQ ID NOS: 1-18 or 55-72 are inserted into theframework region utilizing Kabat numbering.

In one aspect, provided herein is an antibody, or antigen-bindingfragment thereof, that specifically binds to hepcidin, prepared byinjecting a rodent (i.e., mouse, rat or rabbit) with a peptide having anamino acid sequence of any one of SEQ ID NOS: 19-27. In anotherembodiment, the peptide is conjugated to a carrier (e.g., keyhole limpethemocyanin (KLH)) or an adjuvant (complete Freund's adjuvant (CFA) orincomplete Freund's adjuvant (IFA)). In one embodiment, the antibody, orantigen-binding fragment thereof, that specifically binds to amino acidresidues 1-9 of hepcidin. In another embodiment, provided herein is anantibody, or antigen-binding fragment thereof, that specifically bindsto amino acid residues 1-7 of hepcidin.

A hepcidin peptide to which an antibody, or antigen-binding fragmentthereof, binds may have an amino acid sequence of SEQ ID NO: 19.

Provided herein is an antibody, or antigen-binding fragment thereof,that specifically binds to an epitope comprising amino acid sequence ofany one of Hep-5, Hep-9, Hep-20, Hep 22 and Hep-25 where the sequencesof the peptides are provided in the sequence listing.

In one embodiment, the antibody, or antigen-binding fragment thereof,specifically binds to an epitope comprising an amino acid sequence ofHep-20 (SEQ ID NO: 22), Hep-22 (SEQ ID NO: 23) and Hep-25 (SEQ ID NO:19).

In one embodiment, the antibody, or antigen-binding fragment thereof,specifically binds to an epitope comprising Hep-5 (SEQ ID NO: 25) orHep-9 (SEQ ID NO: 24). In another embodiment, provided herein is anantibody, or antigen-binding fragment thereof, that specifically bindsto an epitope comprising amino acid residues 1-9 of hepcidin. In anotherembodiment, the antibody, or antigen-binding fragment thereof,specifically binds to 2, 3, 4, 5, 6, 7, 8, or 9 amino acid residues ofan epitope comprising amino acid residues 1-9 of hepcidin.

In another embodiment, the antibody, or antigen-binding fragmentthereof, is monoclonal antibody comprising a heavy chain CDR1 encoded bySEQ ID NO: 55, a heavy CDR2 encoded by SEQ ID NO: 58, a heavy chain CDR3encoded by SEQ ID NO: 61, a light chain CDR1 encoded by SEQ ID NO: 64, alight CDR2 encoded by SEQ ID NO: 67, and a light chain CDR3 encoded bySEQ ID NO: 70.

In another embodiment, the antibody, or antigen-binding fragmentthereof, is monoclonal antibody comprising a heavy chain CDR1 encoded bySEQ ID NO: 56, a heavy CDR2 encoded by SEQ ID NO: 59, a heavy chain CDR3encoded by SEQ ID NO: 61, a light chain CDR1 encoded by SEQ ID NO: 65, alight CDR2 encoded by SEQ ID NO: 68, and a light chain CDR3 encoded bySEQ ID NO: 71.

In another embodiment, the antibody, or antigen-binding fragmentthereof, is monoclonal antibody comprising a heavy chain CDR1 encoded bySEQ ID NO: 57, a heavy CDR2 encoded by SEQ ID NO: 60, a heavy chain CDR3encoded by SEQ ID NO: 63, a light chain CDR1 encoded by SEQ ID NO: 66, alight CDR2 encoded by SEQ ID NO: 69, and a light chain CDR3 encoded bySEQ ID NO: 72.

The antibody may be, for example, a monoclonal antibody, a chimericantibody, a human antibody, or a humanized antibody. In one embodiment,a humanized variable heavy chain comprises an amino acid sequence setforth as SEQ ID NO: 40. In another embodiment, a humanized variablelight chain comprises an amino acid sequence set forth as SEQ ID NO: 38.

In one aspect, provided herein is an antibody, or antigen-bindingfragment thereof, comprises a heavy chain variable region frameworkregion; and a light chain variable region framework region as set forthin the Sequence Listing below where the CDRs identified in any one ofSEQ ID NOS: 1-18 of 55-72 are inserted into the framework regionutilizing Kabat numbering.

The antigen-binding fragment may be, for example, a Fab fragment, a Fab′fragment, a F(ab′)₂ fragment, an Fv fragment, an scFv fragment, a singlechain binding polypeptide, a Fd fragment, a variable heavy chain, avariable light chain, a dAb fragment or any other type of fragmentdescribed herein. An antigen-binding fragment may be, for example, anAVIMER, a diabody, or a heavy chain dimer. A heavy chain dimer may be,for example, a camelid or a shark heavy chain construct.

An antibody, or antigen-binding fragment thereof, described herein mayhave a dissociation constant (Kd) of about 1 to about 10 pM, from about10 to about 20 pM, from about 1 to about 29 pM, from about 30 to about40 pM, from about 10 to about 100 pM, or from about 20 to about 500 pM.

An antibody, or antigen-binding fragment thereof, described herein mayhave a dissociation constant (Kd) of less than about 500 pM, less thanabout 400 pM, less than about 300 pM, less than about 200 pM, less thanabout 100 pM, less than about 75 pM, less than about 50 pM, less thanabout 30 pM, less than about 25 pM, less than about 20 pM, less thanabout 18 pM, less than about 15 pM, less than about 10 pM, less thanabout 75. pM, less than about 5 pM, less than about 2.5 pM, or less thanabout 1 pM.

An antibody, or antigen-binding fragment thereof, described herein mayhave an affinity for hepcidin or a hepcidin peptide of from about 10⁻⁹to about 10⁻¹⁴, from about 10⁻¹⁰ to about 10⁻¹⁴, from about 10⁻¹¹ toabout 10⁻¹⁴, from about 10⁻¹² to about 10⁻¹⁴, from about 10⁻¹³ to about10⁻¹⁴, from about 10⁻¹⁰ to about 10⁻¹¹, from about 10⁻¹¹ to about 10⁻¹²,from about 10⁻¹² to about 10⁻¹³, or 10⁻¹³ to about 10⁻¹⁴.

Provided herein is a composition, comprising an antibody, orantigen-binding fragment, and an acceptable carrier or excipient.Compositions are described in more detail below.

Also provided herein is an isolated nucleic acid molecule comprising anucleotide sequence that encodes an antibody, or antigen-bindingfragment thereof, described herein. Also provided herein is anexpression vector comprising the nucleic acid molecule, operably linkedto a regulatory control sequence. Also provided herein is a host cellcomprising a vector or a nucleic acid molecule provided herein. Alsoprovided herein is a method of using the host cell to produce anantibody, comprising culturing the host cell under suitable conditionssuch that the nucleic acid is expressed to produce the antibody.

Compositions

Each of the compounds described herein can be used as a composition whencombined with an acceptable carrier or excipient. Such compositions areuseful for in vitro or in vivo analysis or for administration to asubject in vivo or ex vivo for treating a subject with the disclosedcompounds.

Thus pharmaceutical compositions can include, in addition to activeingredient, a pharmaceutically acceptable excipient, carrier, buffer,stabilizer or other materials well known to those skilled in the art.Such materials should be non-toxic and should not interfere with theefficacy of the active ingredient. The precise nature of the carrier orother material will depend on the route of administration.

Pharmaceutical formulations comprising a protein of interest, e.g., anantibody or antigen-binding fragment, identified by the methodsdescribed herein can be prepared for storage by mixing the proteinhaving the desired degree of purity with optional physiologicallyacceptable carriers, excipients or stabilizers (Remington'sPharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the formof lyophilized formulations or aqueous solutions. Acceptable carriers,excipients, or stabilizers are those that are non-toxic to recipients atthe dosages and concentrations employed, and include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN®, PLURONICS® or polyethylene glycol (PEG).

Acceptable carriers are physiologically acceptable to the administeredsubject and retain the therapeutic properties of the compounds with/inwhich it is administered. Acceptable carriers and their formulations areand generally described in, for example, Remington' pharmaceuticalSciences (18th Edition, ed. A. Gennaro, Mack Publishing Co., Easton, Pa.1990). One exemplary carrier is physiological saline. The phrase“pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the subject compoundsfrom the administration site of one organ, or portion of the body, toanother organ, or portion of the body, or in an in vitro assay system.Each carrier is acceptable in the sense of being compatible with theother ingredients of the formulation and not injurious to a subject towhom it is administered. Nor should an acceptable carrier alter thespecific activity of the subject compounds.

In one aspect, provided herein are pharmaceutically acceptable orphysiologically acceptable compositions including solvents (aqueous ornon-aqueous), solutions, emulsions, dispersion media, coatings, isotonicand absorption promoting or delaying agents, compatible withpharmaceutical administration. Pharmaceutical compositions orpharmaceutical formulations therefore refer to a composition suitablefor pharmaceutical use in a subject. The pharmaceutical compositions andformulations include an amount of a compound described herein and apharmaceutically or physiologically acceptable carrier.

Compositions can be formulated to be compatible with a particular routeof administration (i.e., systemic or local). Thus, compositions includecarriers, diluents, or excipients suitable for administration by variousroutes.

In another embodiment, the compositions can further comprise, if needed,an acceptable additive in order to improve the stability of thecompounds in composition and/or to control the release rate of thecomposition. Acceptable additives do not alter the specific activity ofthe subject compounds. Exemplary acceptable additives include, but arenot limited to, a sugar such as mannitol, sorbitol, glucose, xylitol,trehalose, sorbose, sucrose, galactose, dextran, dextrose, fructose,lactose and mixtures thereof. Acceptable additives can be combined withacceptable carriers and/or excipients such as dextrose. Alternatively,exemplary acceptable additives include, but are not limited to, asurfactant such as polysorbate 20 or polysorbate 80 to increasestability of the peptide and decrease gelling of the solution. Thesurfactant can be added to the composition in an amount of 0.01% to 5%of the solution. Addition of such acceptable additives increases thestability and half-life of the composition in storage.

In one embodiment, a composition may contain an isotonic buffer such asa phosphate, acetate, or TRIS buffer in combination with a tonicityagent such as a polyol, Sorbitol, sucrose or sodium chloride, whichtonicifies and stabilizes. A tonicity agent may be present in thecomposition in an amount of about 5%.

In another embodiment, the composition may include a surfactant such asto prevent aggregation and for stabilization at 0.01 to 0.02% wt/vol.

In another embodiment, the pH of the composition may range from 4.5-6.5or 4.5-5.5.

Other exemplary descriptions of pharmaceutical compositions forantibodies may be found in, for example, US 2003/0113316 and U.S. Pat.No. 6,171,586, each incorporated herein by reference in its entirety.

A composition herein may also contain more than one active compound asnecessary for the particular indication being treated, such as thosewith complementary activities that do not adversely affect each other.For example, a method of treatment may further provide animmunosuppressive agent. Such molecules are suitably present incombination in amounts that are effective for the purpose intended.

Active ingredients may be entrapped in microcapsule prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsule andpoly-(methylmethacylate) microcapsule, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980).

Suspensions and crystal forms of antibodies are also contemplatedherein; methods to make suspensions and crystal forms are known to oneof skill in the art.

A composition to be used for in vivo administration must be sterile. Insome embodiments, the compositions of the invention may be sterilized byconventional, well known sterilization techniques. For example,sterilization may be readily accomplished by filtration through sterilefiltration membranes. The resulting solutions may be packaged for use orfiltered under aseptic conditions and lyophilized, the lyophilizedpreparation being combined with a sterile solution prior toadministration.

Freeze-drying may be employed to stabilize polypeptides for long-termstorage, such as when a polypeptide is relatively unstable in liquidcompositions. A lyophilization cycle is usually composed of three steps:freezing, primary drying, and secondary drying; Williams and Polli,Journal of Parenteral Science and Technology, Volume 38, Number 2, pages48-59 (1984). In the freezing step, the solution is cooled until it isadequately frozen. Bulk water in the solution forms ice at this stage.The ice sublimes in the primary drying stage, which is conducted byreducing chamber pressure below the vapor pressure of the ice, using avacuum. Finally, sorbed or bound water is removed at the secondarydrying stage under reduced chamber pressure and an elevated shelftemperature. The process produces a material known as a lyophilizedcake. Thereafter the cake can be reconstituted prior to use. Standardreconstitution practice for lyophilized material is to add back a volumeof pure water (typically equivalent to the volume removed duringlyophilization), although dilute solutions of antibacterial agents aresometimes used in the production of pharmaceuticals for parenteraladministration; Chen, Drug Development and Industrial Pharmacy, Volume18, Numbers 11 and 12, pages 1311-1354 (1992).

Some excipients such as, for example, polyols (including mannitol,sorbitol and glycerol); sugars (including glucose and sucrose); andamino acids (including alanine, glycine and glutamic acid), may act asstabilizers for freeze-dried products; see, e.g., Carpenter et al.,Developments in Biological Standardization, Volume 74, pages 225-239(1991). Polyols and sugars may also be used to protect polypeptides fromfreezing and drying-induced damage and to enhance the stability duringstorage in the dried state. Sugars may be effective in both thefreeze-drying process and during storage. Other classes of molecules,including mono- and disaccharides and polymers such as PVP, have alsobeen reported as stabilizers of lyophilized products.

For injection, a composition and/or medicament may be a powder suitablefor reconstitution with an appropriate solution as described above.Examples of these include, but are not limited to, freeze dried, rotarydried or spray dried powders, amorphous powders, granules, precipitates,or particulates. For injection, the compositions may optionally containstabilizers, pH modifiers, surfactants, bioavailability modifiers andcombinations of these.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsule. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(see, e.g., U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid andy ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the Lupron Depot™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. While encapsulated antibodies remainin the body for a long time, they may denature or aggregate as a resultof exposure to moisture at 37° C., resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS—S bond formation through thio-disulfide interchange, stabilization maybe achieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

A composition described herein may be designed to be short-acting,fast-releasing, long-acting, or sustained-releasing as described herein.In one embodiment, the composition may be formulated for controlledrelease or for slow release.

The pharmaceutical composition can be administered, for example, byinjection, including, but not limited to, subcutaneous, intravitreal,intradermal, intravenous, intra-arterial, intraperitoneal,intracerebreospinal, or intramuscular injection. Excipients and carriersfor use in formulation of compositions for each type of injection arecontemplated herein. The following descriptions are by example only andare not meant to limit the scope of the compositions. Compositions forinjection include, but are not limited to, aqueous solutions (wherewater soluble) or dispersions, as well as sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersion. For intravenous administration, suitable carriers includephysiological saline, bacteriostatic water, Cremophor EL™ (BASF,Parsippany, N.J.) or phosphate buffered saline (PBS). The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol, and liquidpolyetheylene glycol, and the like), and suitable mixtures thereof.Fluidity can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. Antibacterial andantifungal agents include, for example, parabens, chlorobutanol, phenol,ascorbic acid and thimerosal. Isotonic agents, for example, sugars,polyalcohols such as manitol, sorbitol, and sodium chloride may beincluded in the composition. The resulting solutions can be packaged foruse as is, or lyophilized; the lyophilized preparation can later becombined with a sterile solution prior to administration. Forintravenous, injection, or injection at the site of affliction, theactive ingredient will be in the form of a parenterally acceptableaqueous solution which is pyrogen-free and has suitable pH, isotonicityand stability. Those of relevant skill in the art are well able toprepare suitable solutions using, for example, isotonic vehicles such asSodium Chloride Injection, Ringer's Injection, Lactated Ringer'sInjection. Preservatives, stabilizers, buffers, antioxidants and/orother additives may be included, as needed. Sterile injectable solutionscan be prepared by incorporating an active ingredient in the requiredamount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive ingredient into a sterile vehicle which contains a basicdispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Compositions can be conventionally administered intravitreally,sub-cutaneous, or via intravitreal implant.

Compositions can be conventionally administered intravenously, such asby injection of a unit dose, for example. For injection, an activeingredient can be in the form of a parenterally acceptable aqueoussolution which is substantially pyrogen-free and has suitable pH,isotonicity and stability. One can prepare suitable solutions using, forexample, isotonic vehicles such as Sodium Chloride Injection, Ringer'sInjection, Lactated Ringer's Injection. Preservatives, stabilizers,buffers, antioxidants and/or other additives may be included, asrequired. Additionally, compositions can be administered viaaerosolization. (Lahn et al., Aerosolized Anti-T-cell-ReceptorAntibodies Are Effective against Airway Inflammation andHyperreactivity, Int. Arch. Allergy Immuno., 134: 49-55 (2004)).

In one embodiment, the composition is lyophilized, for example, toincrease shelf-life in storage. When the compositions are considered foruse in medicaments or any of the methods provided herein, it iscontemplated that the composition can be substantially free of pyrogenssuch that the composition will not cause an inflammatory reaction or anunsafe allergic reaction when administered to a human subject. Testingcompositions for pyrogens and preparing compositions substantially freeof pyrogens are well understood to one or ordinary skill of the art andcan be accomplished using commercially available kits.

Acceptable carriers can contain a compound that stabilizes, increases ordelays absorption or clearance. Such compounds include, for example,carbohydrates, such as glucose, sucrose, or dextrans; low molecularweight proteins; compositions that reduce the clearance or hydrolysis ofpeptides; or excipients or other stabilizers and/or buffers. Agents thatdelay absorption include, for example, aluminum monostearate andgelatin. Detergents can also be used to stabilize or to increase ordecrease the absorption of the pharmaceutical composition, includingliposomal carriers. To protect from digestion the compound can becomplexed with a composition to render it resistant to acidic andenzymatic hydrolysis, or the compound can be complexed in anappropriately resistant carrier such as a liposome. Means of protectingcompounds from digestion are known in the art (see, e.g., Fix (1996)Pharm Res. 13:1760 1764; Samanen (1996) J. Pharm. Pharmacol. 48:119 135;and U.S. Pat. No. 5,391,377, describing lipid compositions for oraldelivery of therapeutic agents).

The phrase “pharmaceutically acceptable” refers to molecular entitiesand compositions that are physiologically tolerable and do not typicallyproduce an allergic or similar untoward reaction, such as gastric upset,dizziness and the like, when administered to a human.

The term “unit dose” when used in reference to a therapeutic compositionrefers to physically discrete units suitable as unitary dosage forhumans, each unit containing a predetermined quantity of active materialcalculated to produce the desired therapeutic effect in association withthe required diluent; i.e., carrier, or vehicle.

The compositions can be administered in a manner compatible with thedosage formulation, and in a therapeutically effective amount. Thequantity to be administered depends on the subject to be treated,capacity of the subject's immune system to utilize the activeingredient, and degree of binding capacity desired. Precise amounts ofactive ingredient required to be administered depend on the judgment ofthe practitioner and are peculiar to each individual. Suitable regimesfor initial administration and booster shots are also variable, but aretypified by an initial administration followed by repeated doses at oneor more hour intervals by a subsequent injection or otheradministration. Alternatively, continuous intravenous infusion that issufficient to maintain concentrations in the blood are contemplated.

One embodiment contemplates the use of the compositions described hereinto make a medicament for treating a condition, disease or disorderdescribed herein. Medicaments can be formulated based on the physicalcharacteristics of the subject needing treatment, and can be formulatedin single or multiple formulations based on the stage of the condition,disease or disorder. Medicaments can be packaged in a suitable packagewith appropriate labels for the distribution to hospitals and clinicswherein the label is for the indication of treating a subject having adisease described herein. Medicaments can be packaged as a single ormultiple units. Instructions for the dosage and administration of thecompositions can be included with the packages as described below. Theinvention is further directed to medicaments of an anti-hepcidinantibody or antigen binding fragment thereof described hereinabove and apharmaceutically acceptable carrier.

Provided herein are compositions of antibodies and antigen-bindingfragments thereof that bind hepcidin and include those such as describedelsewhere herein. Antibodies and antigen-binding fragments thereof thatbind hepcidin as described herein can be used for the treatment ofvarious diseases and conditions associated with iron homeostasis.

A composition (an antibody or an antigen-binding fragment describedherein) can be administered alone or in combination with a secondcomposition either simultaneously or sequentially dependent upon thecondition to be treated. In one embodiment, a second therapeutictreatment is an erythropoiesis stimulator. When two or more compositionsare administered, the compositions can be administered in combination(either sequentially or simultaneously). A composition can beadministered in a single dose or multiple doses.

When formulated for administration to human subjects, the compositionsmay be formulated to be free of pyrogens. Testing compositions forpyrogens and preparing pharmaceutical compositions free of pyrogens arewell understood to one of ordinary skill in the art.

One embodiment contemplates the use of any of the compositions of thepresent invention to make a medicament for treating a disorder of thepresent invention. Medicaments can be formulated based on the physicalcharacteristics of the subject needing treatment, and can be formulatedin single or multiple formulations based on the disorder. Medicaments ofthe present invention can be packaged in a suitable pharmaceuticalpackage with appropriate labels for the distribution to hospitals andclinics wherein the label is for the indication of treating a disorderas described herein in a subject. Medicaments can be packaged as asingle or multiple units. Instructions for the dosage and administrationof the pharmaceutical compositions of the present invention can beincluded with the pharmaceutical packages.

Diagnostics

Provided herein is a method of diagnosing a hepcidin-related disorder,comprising: (a) contacting a biological sample from a subject suspectedof having said disorder with an antibody, or antigen-binding fragmentthereof, described herein under conditions that allow binding of theantibody or antigen-binding fragment thereof, to hepcidin; and (b)detecting and/or quantitating the hepcidin bound to the antibody, orantigen-binding fragment thereof, wherein the amount of hepcidin in thesample, as quantitated in (b), above a threshold level indicates thepresence of hepcidin-related disorder and below the threshold levelindicates the absence of hepcidin-related disorder.

A method of differentiating an inflammatory disease from anon-inflammatory disease, comprising: (a) contacting a biological samplefrom a human suspected of having said disorder with an antibody orantigen-binding fragment thereof, described herein under conditions thatallow binding of the antibody or antigen-binding fragment thereof, tohepcidin; and (b) detecting and/or quantitating the hepcidin bound tothe antibody or antigen-binding fragment thereof, wherein the amount ofhepcidin, as quantitated in (b), above a threshold level indicates thepresence of inflammatory disease and below the threshold level indicatesthe absence of inflammatory disease.

In one embodiment, the antibody or antigen-binding fragment furthercomprises a detectable moiety. Detection can occur in vitro, in vivo orex vivo. In vitro assays for the detection and/or determination(quantification, qualification, etc.) of hepcidin with the antibodies orantigen-binding fragments thereof include but are not limited to, forexample, ELISAs, RIAs and western blots. In vitro detection, diagnosisor monitoring of hepcidin can occur by obtaining a sample (e.g., a bloodsample) from a subject and testing the sample in, for example, astandard ELISA assay. For example, a 96-well microtiter plate can becoated with an antibody or antigen-binding fragment thereof describedherein, washed and coating with PBS-Tween/BSA to inhibit non-specificbinding. The blood sample can be serially diluted and placed in singleor duplicate wells compared to a serially-diluted standard curve ofhepcidin. After incubating and washing the wells, an anti-hepcidinantibody labeled with biotin can be added, followed by addition ofstreptavidin-alkaline phosphatase. The wells can be washed and asubstrate (horseradish peroxidase) added to develop the plate. The platecan be read using a conventional plate reader and software.

When detection occurs in vivo, contacting occurs via administration ofthe antibody or antigen binding fragment using any conventional meanssuch as those described elsewhere herein. In such methods, detection ofhepcidin in a sample or a subject can be used to diagnose a disease ordisorder associated with, or correlated with the activity of such asthose diseases and disorders described herein.

In the in vivo detection, diagnosis or monitoring of hepcidin, a subjectis administered an antibody or antigen-binding fragment that binds tohepcidin, which antibody or antigen-binding fragment is bound to adetectable moiety. The detectable moiety can be visualized usingart-recognized methods such as, but not limited to, magnetic resonanceimaging (MRI), fluorescence, radioimaging, light sources supplied byendoscopes, laparoscopes, or intravascular catheter (i.e., via detectionof photoactive agents), photoscanning, positron emission tomography(PET) scanning, whole body nuclear magnetic resonance (NMR),radioscintography, single photon emission computed tomography (SPECT),targeted near infrared region (NIR) scanning, X-ray, ultrasound, etc.such as described, for example, in U.S. Pat. No. 6,096,289, U.S. Pat.No. 7,115,716, U.S. Pat. No. 7,112,412, U.S. Patent Application No.20030003048 and U. S. Patent Application No. 20060147379, each of whichis incorporated herein in its entirety by reference. Labels fordetecting compounds using such methods are also known in the art anddescribed in such patents and applications and are incorporated hereinby reference. Visualization of the detectable moiety can allow fordetection, diagnosis, and/or monitoring of a condition or diseaseassociated with hepcidin.

Additional diagnostic assays that utilize antibodies specific to thedesired target protein, i.e., hepcidin, are known in the art and arealso contemplated herein.

For in vitro detection methods, samples to be obtained from a subjectinclude, but are not limited to, blood, tissue biopsy samples and fluidtherefrom.

Thus, the present invention provides humanized antibodies andantigen-binding fragments thereof against hepcidin which are useful fordetecting or diagnosing levels of hepcidin associated with a disease ordisorder, potentially indicating need for therapeutic treatment. Incertain embodiments, the antibodies comprise a humanized anti-hepcidinantibody described herein. In other embodiments the antibody furthercomprises a second agent. Such an agent can be a molecule or moiety suchas, for example, a reporter molecule or a detectable label. Detectablelabels/moieties for such detection methods are known in the art and aredescribed in more detail below. Reporter molecules are any moiety whichcan be detected using an assay. Non-limiting examples of reportermolecules which have been conjugated to polypeptides include enzymes,radiolabels, haptens, fluorescent labels, phosphorescent molecules,chemiluminescent molecules, chromophores, luminescent molecules,photoaffinity molecules, colored particles or ligands, such as biotin.Detectable labels include compounds and/or elements that can be detecteddue to their specific functional properties, and/or chemicalcharacteristics, the use of which allows the polypeptide to which theyare attached to be detected, and/or further quantified if desired. Manyappropriate detectable (imaging) agents are known in the art, as aremethods for their attachment to polypeptides (see, for e.g., U.S. Pat.Nos. 5,021,236; 4,938,948; and 4,472,509, each of which is herebyincorporated by reference).

Methods of joining polypeptides such as antibodies with detectablemoieties are known in the art and include, for example, recombinant DNAtechnology to form fusion proteins and conjugation (e.g., chemicalconjugation). Methods for preparing fusion proteins by chemicalconjugation or recombinant engineering are well-known in the art.Methods of covalently and non-covalently linking components are alsoknown in the art. See, e.g., Williams (1995) Biochemistry 34:1787 1797;Dobeli (1998) Protein Expr. Purif. 12:404-414; and Kroll (1993) DNACell. Biol. 12: 441-453.

It may be necessary, in some instances, to introduce an unstructuredpolypeptide linker region between a label or a moiety and one or moreportion of the antibodies, antigen-binding fragments or binding proteinsdescribed herein. A linker can facilitate enhanced flexibility, and/orreduce steric hindrance between any two fragments. The linker can alsofacilitate the appropriate folding of each fragment to occur. The linkercan be of natural origin, such as a sequence determined to exist inrandom coil between two domains of a protein. One linker sequence is thelinker found between the C-terminal and N-terminal domains of the RNApolymerase a subunit. Other examples of naturally occurring linkersinclude linkers found in the 1CI and LexA proteins.

Within a linker, an amino acid sequence can be varied based on thecharacteristics of the linker as determined empirically or as revealedby modeling. Considerations in choosing a linker include flexibility ofthe linker, charge of the linker, and presence of some amino acids ofthe linker in the naturally-occurring subunits. The linker can also bedesigned such that residues in the linker contact deoxyribose nucleicacid (DNA), thereby influencing binding affinity or specificity, or tointeract with other proteins. In some cases, such as when it isnecessary to span a longer distance between subunits or when the domainsmust be held in a particular configuration, the linker can, optionally,contain an additional folded domain. In some embodiments, the design ofa linker can involve an arrangement of domains which requires the linkerto span a relatively short distance, e.g., less than about 10 Angstroms(Å). However, in certain embodiments, linkers span a distance of up toabout 50 Angstroms.

Within the linker, the amino acid sequence can be varied based on thecharacteristics of the linker as determined empirically or as revealedby modeling. Considerations in choosing a linker include flexibility ofthe linker, charge of the linker, and presence of some amino acids ofthe linker in the naturally-occurring subunits. The linker can also bedesigned such that residues in the linker contact DNA, therebyinfluencing binding affinity or specificity, or to interact with otherproteins. In some cases, when it is necessary to span a longer distancebetween subunits or when the domains must be held in a particularconfiguration, the linker can optionally contain an additional foldeddomain.

Methods for coupling polypeptides (free or cell-bound) to beads areknown in the art. Methods for selecting coupled polypeptides or cellsdisplaying a polypeptide are also known in the art. Briefly,paramagnetic polystyrene microparticles are commercially available(Spherotech, Inc., Libertyville, Ill.; Invitrogen, Carlsbad, Calif.)that couple peptides to microparticle surfaces that have been modifiedwith functional groups or coated with various antibodies or ligands suchas, for example, avidin, streptavidin or biotin.

The paramagnetic property of microparticles allows them to be separatedfrom solution using a magnet. The microparticles can be easilyre-suspended when removed from the magnet. Polypeptides can be coupledto paramagnetic polystyrene microparticles coated with a polyurethanelayer in a tube. The hydroxy groups on the microparticle surface areactivated by reaction with p-toluensulphonyl chloride (Nilsson K andMosbach K. “p-Toluenesulfonyl chloride as an activating agent of agarosefor the preparation of immobilized affinity ligands and proteins.” Eur.J. Biochem. 1980:112: 397-402). Alternatively, paramagnetic polystyrenemicroparticles containing surface carboxylic acid can be activated witha carbodiimide followed by coupling to a polypeptide, resulting in astable amide bond between a primary amino group of the polypeptide andthe carboxylic acid groups on the surface of the microparticles(Nakajima N and Ikade Y, Mechanism of amide formation by carbodiimidefor bioconjugation in aqueous media, Bioconjugate Chem. 1995, 6(1):123-130; Gilles M A, Hudson A Q and Borders C L Jr, Stability ofwater-soluble carbodiimides in aqueous solution, Anal Biochem. 1990 Feb.1; 184(2):244-248; Sehgal D and Vijay I K, a method for the highefficiency of water-soluble carbodiimide-mediated amidation, AnalBiochem. 1994 April; 218(1):87-91; Szajani B et al, Effects ofcarbodiimide structure on the immobilization of enzymes, Appl BiochemBiotechnol. 1991 August; 30(2): 225-231). Another option is to couplebiotinylated polypeptides to paramagnetic polystyrene microparticleswhose surfaces have been covalently linked with a monolayer ofstreptavidin. (Argarana C E, Kuntz I D, Birken S, Axel R, Cantor C R.Molecular cloning and nucleotide sequence of the streptavidin gene.Nucleic Acids Res. 1986; 14(4):1871-82; Pahler A, Hendrickson W A,Gawinowicz Kolks M A, Aragana C E, Cantor C R. Characterization andcrystallization of core streptavidin. J Biol Chem1987:262(29):13933-13937).

Polypeptides can be conjugated to a wide variety of fluorescent dyes,quenchers and haptens such as fluorescein, R-phycoerythrin, and biotin.Conjugation can occur either during polypeptide synthesis or after thepolypeptide has been synthesized and purified. Biotin is a small (244kilodaltons) vitamin that binds with high affinity to avidin andstreptavidin proteins and can be conjugated to most peptides withoutaltering their biological activities. Biotin-labeled polypeptides areeasily purified from unlabeled polypeptides using immobilizedstreptavidin and avidin affinity gels, and streptavidin oravidin-conjugated probes can be used to detect biotinylated polypeptidesin, for example, ELISA, dot blot or Western blot applications.N-hydroxysuccinimide esters of biotin are the most commonly used type ofbiotinylation agent. N-hydroxysuccinimide-activated biotins reactefficiently with primary amino groups in physiological buffers to formstable amide bonds. Polypeptides have primary amines at the N-terminusand can also have several primary amines in the side chain of lysineresidues that are available as targets for labeling withN-hydroxysuccinimide-activated biotin reagents. Several differentN-hydroxysuccinimide esters of biotin are available, with varyingproperties and spacer arm length (Pierce, Rockford, Ill.). Thesulfo-N-hydroxysuccinimide ester reagents are water soluble, enablingreactions to be performed in the absence of organic solvents.

The mole-to-mole ratio of biotin to polypeptide can be estimated using a2-(4′-Hydroxyazobenzene-2-carboxylic acid) assay using art-recognizedtechniques (Green, N M, (1975) “Avidin. In Advances in ProteinChemistry.” Academic Press, New York. 29, 85-133; Green, N M, (1971)“The use of bifunctional biotinyl compounds to determine the arrangementof subunits in avidin.” Biochem J. 125, 781-791; Green, N M., (1965) “Aspectrophotometric assay for avidin and biotin based on binding of dyesby avidin.” Biochem. J. 94: 23c-24c). Several biotin molecules can beconjugated to a polypeptide and each biotin molecule can bind onemolecule of avidin. The biotin-avidin bond formation is very rapid andstable in organic solvents, extreme pH and denaturing reagents. Toquantitate biotinylation, a solution containing the biotinylatedpolypeptide is added to a mixture of2-(4′-Hydroxyazobenzene-2-carboxylic acid) and avidin. Because biotinhas a higher affinity for avidin, it displaces the2-(4′-Hydroxyazobenzene-2-carboxylic acid) and the absorbance at 500nanometers decreases proportionately. The amount of biotin in a solutioncan be quantitated in a single cuvette by measuring the absorbance ofthe 2-(4′-Hydroxyazobenzene-2-carboxylic acid)-avidin solution beforeand after addition of the biotin-containing peptide. The change inabsorbance relates to the amount of biotin in the sample by theextinction coefficient of the 2-(4′-Hydroxyazobenzene-2-carboxylicacid)-avidin complex.

Alternatively, an antibody, antigen-binding fragment or binding proteincan be conjugated with a fluorescent moiety Conjugating polypeptideswith fluorescent moieties (e.g., R-Phycoerythrin, fluoresceinisothiocyanate (FITC), etc.) can be accomplished using art-recognizedtechniques described in, for example, Glazer, A N and Stryer L. (1984).Trends Biochem. Sci. 9:423-7; Kronick, M N and Grossman, P D (1983)Clin. Chem. 29:1582-6; Lanier, L L and Loken, M R (1984) J. Immunol.,132:151-156; Parks, D R et al. (1984) Cytometry 5:159-68; Hardy, R R etal. (1983) Nature 306:270-2; Hardy R R et al. (1984) J. Exp. Med.159:1169-88; Kronick, M N (1986) J. Immuno. Meth. 92:1-13; Der-Balian G,Kameda, N and Rowley, G. (1988) Anal. Biochem. 173:59-63.

In one non-limiting embodiment, an antibody antigen-binding fragment canbe associated with (conjugated to) a detectable label, such as aradionuclide, iron-related compound, a dye, an imaging agent or afluorescent agent for immunodetection of hepcidin which can be used tovisualize binding of the antibodies to hepcidin in vitro and/or in vivo.

Non-limiting examples of radiolabels include, for example, ³²P, ³³P,⁴³K, ⁵²Fe, ⁵⁷Co, ⁶⁴Cu, ⁶⁷Ga, ⁶⁷Cu, ⁶⁸Ga, ⁷¹Ge, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br, ⁷⁷As,⁷⁷Br, ⁸¹Rb/⁸¹MKr, ⁸⁷MSr, ⁹⁰Y, ⁹⁷Ru, ⁹⁹Tc, ¹⁰⁰Pd, ¹⁰¹Rh, ¹⁰³Pb, ¹⁰⁵Rh,¹⁰⁹Pd, ¹¹¹Ag, ¹¹¹In, ¹¹³In, ¹¹⁹Sb, ¹²¹Sn, ¹²³I, ¹²⁵I, ¹²⁷Cs, ¹²⁸Ba,¹²⁹Cs, ¹³¹I, ¹³¹Cs, ¹⁴³Pr, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁶⁹Eu, ¹⁷⁷Lu, ¹⁸⁶Re,¹⁸⁸Re, ¹⁸⁹Re, ¹⁹¹Os, ¹⁹³Pt, ¹⁹⁴Ir, ¹⁹⁷Hg, ¹⁹⁹Au, ²⁰³Pb, ²¹¹At, ²¹²Pb,²¹²Bi and ²¹³Bi. Radiolabels can be attached to compounds usingconventional chemistry known in the art of antibody imaging.Radiolabeled compounds are useful in in vitro diagnostics techniques andin in vivo radioimaging techniques and in radioimmunotherapy.

In one embodiment, the antibody or antigen-binding fragment thereof canbe conjugated to both a therapeutic moiety and a detectable moiety. Anantibody or antigen-binding fragment thereof can be conjugated to, orrecombinantly engineered with, an affinity tag (e.g., a purificationtag). Affinity tags such as, for example, His6 tags (SEQ ID NO: 28) areconventional in the art.

Antibodies or antigen-binding fragments thereof provided herein are suchthat they can be conjugated or linked to a therapeutic moiety and/or animaging or a detectable moiety and/or an affinity tag. Methods forconjugating or linking polypeptides are well known in the art.Associations (binding) between compounds and labels include any meansknown in the art including, but not limited to, covalent andnon-covalent interactions, chemical conjugation as well as recombinanttechniques.

Methods of Treatment

Provided herein is a method of inducing a response in a subject (humanor non-human) by administering to the subject a composition of anantibody, or antigen-binding fragment thereof, that binds to hepcidin.The binding site to which the antibody binds can be a continuous orconformation/dis-continuous epitope. In one embodiment, an antibody, orantigen-binding fragment thereof, specifically binds to an epitopecomprising amino acid residues 1-9 of hepcidin. In another embodiment,an antibody, or antigen-binding fragment thereof, specifically binds to2, 3, 4, 5, 6, 7, 8 or 9 amino acid residues of an epitope comprisingamino acid residues 1-9 of hepcidin. In yet another embodiment, anantibody, or antigen-binding fragment thereof, specifically binds toHep-20, Hep-22, and Hep-25.

Hepcidin may have an amino acid sequence of, for example, SEQ ID NO: 19.A hepcidin peptide may have an amino acid sequence of, for example, anyone of SEQ ID NOS: 20-25. In yet another embodiment, an antibody, orantigen-binding fragment thereof, binds to an amino acid sequence setforth in any one of the peptide SEQ ID NOS described herein including,for example, SEQ ID NOS: 19-27.

An effective response of the present invention is achieved when thesubject experiences partial or total alleviation or reduction of signsor symptoms of illness, and specifically includes, without limitation,prolongation of survival. The expected progression-free survival timesmay be measured in months to years, depending on prognostic factorsincluding the number of relapses, stage of disease, and other factors.Prolonging survival includes without limitation times of at least 1month (mo), about at least 2 mos., about at least 3 mos., about at least4 mos., about at least 6 mos., about at least 1 year, about at least 2years, about at least 3 years, etc. Overall survival can be alsomeasured in months to years. Alternatively, an effective response may bethat a subject's symptoms remain static. Further indications oftreatment of indications are described in more detail below.

Compositions of antibodies and antigen-binding fragments describedherein can be used as non-therapeutic agents (e.g., as affinitypurification agents). Generally, in one such embodiment, a protein ofinterest is immobilized on a solid phase such a Sephadex resin or filterpaper, using conventional methods known in the art. The immobilizedprotein is contacted with a sample containing the target of interest (orfragment thereof) to be purified, and thereafter the support is washedwith a suitable solvent that will remove substantially all the materialin the sample except the target protein, which is bound to theimmobilized antibody. Finally, the support is washed with anothersuitable solvent, such as glycine buffer, pH 5.0, which will release thetarget protein. In addition to purification, compositions can be usedfor detection, diagnosis and therapy of diseases and disorders describedherein.

The term “contacting” as used herein refers to adding together asolution or composition of a compound with a liquid medium bathing thepolypeptides, cells, tissue or organ from an organism. Alternately,“contacting” refers to mixing together a solution or composition of acompound, with a liquid such as blood, serum, or plasma derived from anorganism. For in vitro applications, a composition can also compriseanother component, such as dimethyl sulfoxide (DMSO). DMSO facilitatesthe uptake of the compounds or solubility of the compounds. The solutioncomprising the test compound may be added to the medium bathing thecells, tissues, or organs, or mixed with another liquid such as blood,by utilizing a delivery apparatus, such as a pipette-based device orsyringe-based device. For in vivo applications, contacting can occur,for example, via administration of a composition to a subject by anysuitable means; compositions with pharmaceutically acceptable excipientsand carriers have been described in more detail above.

A “subject” (e.g., a mammal such as a human or a non-human animal suchas a primate, rodent, cow, horse, pig, sheep, etc.) according to oneembodiment of the present application, is a mammal who exhibits one ormore clinical manifestations and/or symptoms of a disease or disorderdescribed herein.

A composition described herein may be administered to a subject in atherapeutically effective amount which is effective for producing somedesired therapeutic effect by inhibiting a disease or disorder such asdescribed herein which can be associated with hepcidin, at a reasonablebenefit/risk ratio applicable to any medical treatment. For theadministration of the present compositions to human subjects, thecompositions can be formulated by methodology known by one of ordinaryskill in the art. A therapeutically effective amount is an amount thatachieves at least partially a desired therapeutic or prophylactic effectin an organ or tissue. The amount of an anti-hepcidin antibody orantigen binding fragment thereof necessary to bring about preventionand/or therapeutic treatment of a disease or disorder is not fixed perse. The amount of anti-hepcidin antibody or antigen binding fragmentthereof administered may vary with the type of disease, extensiveness ofthe disease, and size of the mammal suffering from the disease ordisorder. In one embodiment, two or more anti-hepcidin antibodiesdescribed herein are administered to a subject in combination.Combination includes concomitant or subsequent administration of theantibodies.

“Administering” is defined herein as a means providing the compositionto the subject in a manner that results in the composition being insidethe subject's body. Such an administration can be by any routeincluding, without limitation, locally, regionally or systemically bysubcutaneous, intravitreal, intradermal, intravenous, intra-arterial,intraperitoneal, intracerebreospinal, or intramuscular administration(e.g., injection). “Concurrent administration” means administrationwithin a relatively short time period from each other; such time periodcan be less than 2 weeks, less than 7 days, less than 1 day and couldeven be administered simultaneously.

Actual dosage levels of the active ingredients in the compositions canbe varied so as to obtain an amount of the active ingredient that iseffective to achieve the desired therapeutic response for a particularsubject, composition, and mode of administration, without being toxic tothe subject. The selected dosage level will depend upon a variety offactors including the activity of the particular compound employed, theroute of administration, the time of administration, the rate ofexcretion of the particular compound being employed, the duration of thetreatment, other drugs, compounds and/or materials used in combinationwith the particular composition employed, the age, sex, weight,condition, general health and prior medical history of the subject beingtreated, and like factors well known in the medical arts.

The antibodies and antigen-binding fragments described herein may beadministered to a subject in various dosing amounts and over varioustime frames. Non-limiting doses include about 0.01 mg/kg, about 0.05mg/kg, about 0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 5 mg/kg,about 10 mg/kg, about 20 mg/kg, about 30 mg/kg, about 40 mg/kg, about 50mg/kg, about 60 mg/kg, about 70 mg/kg, about 80 mg/kg, about 90 mg/kg,about 100 mg/kg, about 125 mg/kg, about 150 mg/kg, about 175 mg/kg,about 200 mg/kg, or any integer in between. Additionally, the dose(s) ofan antibody or antigen-binding fragment can be administered twice aweek, weekly, every two weeks, every three weeks, every 4 weeks, every 6weeks, every 8 weeks, every 12 weeks, or any combination of weekstherein. Dosing cycles are also contemplated such as, for example,administering antibodies or antigen-binding fragments thereof once ortwice a week for 4 weeks, followed by two weeks without therapy.Additional dosing cycles including, for example, different combinationsof the doses and weekly cycles described herein are also contemplatedwithin the invention.

Therapeutically effective amounts of a composition may vary and dependon the severity of the disease and the weight and general state of thesubject being treated, but generally range from about 1.0 μg/kg to about100 mg/kg body weight, or about 10 μg/kg to about 30 mg/kg, or about 0.1mg/kg to about 10 mg/kg or about 1 mg/kg to about 10 mg/kg perapplication. Administration can be daily, on alternating days, weekly,twice a month, monthly or more or less frequently, as necessarydepending on the response to the disorder or condition and the subject'stolerance of the therapy. Maintenance dosages over a longer period oftime, such as 4, 5, 6, 7, 8, 10 or 12 weeks or longer may be neededuntil a desired suppression of disorder symptoms occurs, and dosages maybe adjusted as necessary. The progress of this therapy is easilymonitored by conventional techniques and assays.

Specific dosages may be adjusted depending on conditions of disease, theage, body weight, general health conditions, sex, and diet of thesubject, dose intervals, administration routes, excretion rate, andcombinations of drugs. Any of the above dosage forms containingeffective amounts are well within the bounds of routine experimentationand therefore, well within the scope of the instant invention.

In some embodiments, the specific binding agent or antibody of theinvention is administered intravenously in a physiological solution at adose ranging between 0.01 mg/kg to 100 mg/kg at a frequency ranging fromdaily to weekly to monthly (e.g. every day, every other day, every thirdday, or 2, 3, 4, 5, or 6 times per week), preferably a dose ranging from0.1 to 45 mg/kg, 0.1 to 15 mg/kg or 0.1 to 10 mg/kg at a frequency of 2or 3 times per week, or up to 45 mg/kg once a month.

“Contacting” is defined herein as a means of bringing a composition asprovided herein in physical proximity with a cell, organ, tissue orfluid as described herein. Contacting encompasses systemic or localadministration of any of the compositions provided herein and includes,without limitation, in vitro, in vivo and/or ex vivo procedures andmethods. “Combining” and “contacting” are used interchangeably hereinand are meant to be defined in the same way.

An antibody described herein may be administered by any suitable means,either systemically or locally, including via parenteral, subcutaneous,intraperitoneal, intracerebreospinal, intrapulmonary, and intranasaladministration, and, if desired for local treatment, intralesionaladministration. Parenteral routes include intravenous, intraarterial,intraperitoneal, epidural, intrathecal administration. In addition, thespecific binding agent or antibody is suitably administered by pulseinfusion, particularly with declining doses of the specific bindingagent or antibody. In one embodiment, compositions may be administeredgiven by injection depending in part on whether the administration isbrief or chronic. Other modes of administration methods arecontemplated, including topical, particularly transdermal, transmucosal,rectal, oral or local administration e.g. through a catheter placedclose to the desired site.

A response is achieved when the subject experiences partial or totalalleviation, or reduction of signs or symptoms of illness, andspecifically includes, without limitation, prolongation of survival. Theexpected progression-free survival times can be measured in months toyears, depending on prognostic factors including the number of relapses,stage of disease, and other factors. Prolonging survival includeswithout limitation times of at least 1 month (mo), about at least 2months (mos.), about at least 3 mos., about at least 4 mos., about atleast 6 mos., about at least 1 year, about at least 2 years, about atleast 3 years, or more. Overall survival can also be measured in monthsto years. The subject's symptoms can remain static or can decrease.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount (ED50) of the compositionrequired. For example, the physician or veterinarian could start dosesof the compounds employed in the composition at levels lower than thatrequired in order to achieve the desired therapeutic effect andgradually increase the dosage until the desired effect is achieved.Alternatively, a dose can remain constant.

Compositions can be administered to a subject by any convenient routesuch as described above. Regardless of the route of administrationselected, the compounds of the present invention, which can be used in asuitable hydrated form, and/or the compositions, are formulated intoacceptable dosage forms such as described below or by other conventionalmethods known to those of skill in the art.

Antibodies and/or other agents may be combined in separate compositionsfor simultaneous or sequential administration. In one embodiment,simultaneous administration comprises one or more compositions that areadministered at the same time, or within 30 minutes of each other.Administration may occur at the same or different sites.

Toxicity and therapeutic efficacy of such ingredient can be determinedby standard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.While compounds that exhibit toxic side effects may be used, care shouldbe taken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage to healthycells and, thereby, reduce side effects.

Data obtained from cell culture assays and animal studies can be used informulating a range of dosage for use in humans. The dosage of suchcompounds lies preferably within a range of circulating concentrationsthat include the ED₅₀ with little or no toxicity. The dosage may varywithin this range depending upon the dosage form employed and the routeof administration utilized. For any compound used in the method of theinvention, the therapeutically effective dose can be estimated initiallyfrom cell culture assays. A dose can be formulated in animal models toachieve a circulating plasma concentration arrange that includes theIC₅₀ (i.e., the concentration of the test compound which achieves ahalf-maximal inhibition) as determined in cell culture. Levels in plasmacan be measured, for example, by high performance liquid chromatography.Such information can be used to more accurately determine useful dosesin humans.

As used herein, an antibody, or antigen-binding fragment thereof, may bea hepcidin activity antagonist, meaning a substance that inhibitshepcidin's iron-regulating activity.

In one aspect, the hepcidin activity antagonist can be a substance thatinhibits the function of hepcidin, for example, by inhibiting bindingbetween hepcidin and ferroportin, by inhibiting hepcidin-controlledcellular iron retention, or by facilitating ferroportin dependent irontransport. Hepcidin activity antagonists include antibodies, orantigen-binding fragments thereof, that bind hepcidin and inhibit itsactivity. An antibody, or antigen-binding fragment thereof, may in someinstances, bind to ferroportin but do not activate ferroportin irontransport.

In yet other embodiments, an antibody, or antigen-binding fragmentthereof, described herein may inhibit (or neutralize) hepcidiniron-regulating activity, in vitro and/or also in vivo. Suchhepcidin-neutralizing antibodies are therapeutically useful forhepcidin-related disorders or disorders of iron homeostasis. Hepcidinneutralizing activity may be measured, for example, through a number ofmarkers such as ferritin/iron levels, red blood cell count, red bloodcell characteristics (hemoglobin content and/or cell volume), early redblood cell characteristics (reticulocyte numbers, hemoglobin content orcell volume), ferroportin internalization, or iron transport. In onenon-limiting embodiment, an antibody, or antigen-binding fragmentthereof, described herein decreases intracellular iron concentration atan EC₅₀ of about 10⁻⁸ M or less and/or increases circulating ironconcentration.

An antibody, or antigen-binding fragment thereof, described herein mayantagonize the effect of human hepcidin or inhibit hepcidiniron-regulating activity. In some embodiments, an antibody, orantigen-binding fragment thereof, described herein exerts an effect atan EC₅₀ of about 1×10⁻⁸ M or less, or about 1×10⁻⁷ M or less. Forexample, an antibody may decrease the intracellular iron level in a cellat an EC₅₀ of about 1×10⁻⁸ M or less, or may reduce ferritin expressionat an EC₅₀ of about 1×10⁻⁸ M or less, as determined by a ferritin assay.In other embodiments, an antibody as described herein may reduce freeserum hepcidin levels by at least about 20%, by at least about 30%, byat least about 40%, by at least about 50%, by at least about 60%, by atleast about 70%, by at least about 80%, or by at least about 90%compared to a control antibody or compared to a placebo. In otherembodiments, an antibody as described herein may increase red blood cellcount (number), red blood cell mean cell volume or red blood cellhemoglobin content, increase hemoglobin, increase hematocrit, increase %Tsat, increase circulating (or serum) iron levels, and/or increase ornormalize reticulocyte count, reticulocyte mean cell volume,reticulocyte hemoglobin content or reticulocyte numbers.

Provided herein are diagnostic methods utilizing an antibody orantigen-binding fragment thereof, described herein. An antibody, orantigen-binding fragment thereof, described herein, may also be used forpurification purposes.

Also provided herein are therapeutic methods utilizing an antibody orantigen-binding fragment thereof, described herein. Such antibodies, orantigen-binding fragments thereof, may be used to treat ahepcidin-related disorder. Hepcidin-related disorders, inflammatorydiseases, and diseases or disorders of iron homeostasis for which themethods may be applied include but are not limited to African ironoverload, alpha thalassemia, Alzheimer's disease, anemia, anemia ofcancer, anemia of chronic disease, anemia of inflammation,arteriosclerosis or atherosclerosis (including coronary artery disease,cerebrovascular disease or peripheral occlusive arterial disease),ataxias, ataxias related to iron, atransferrinemia, cancer,ceruloplasmin deficiency, chemotherapy-induced anemia, chronicrenal/kidney disease (stage I, II, III, IV or V), including end stagerenal disease or chronic renal/kidney failure, acute kidney injury(AKI), cirrhosis of liver, classic hemochromatosis, collagen-inducedarthritis (CIA), conditions with hepcidin excess (elevated hepcidin),congenital dyserythropoietic anemia, congestive heart failure, Crohn'sdisease, Celiac disease, inflammatory bowel disease (IBD), diabetes,disorders of iron biodistribution, disorders of iron homeostasis,disorders of iron metabolism, ferroportin disease, ferroportin mutationhemochromatosis, folate deficiency, Friedrich's ataxia, funicularmyelosis, gracile syndrome, H. pylori infection or other bacterialinfections, Hallervordan Spatz disease, hemochromatosis, hemochromatosisresulting from mutations in transferrin receptor 2, hemoglobinopathies,hepatitis, hepatitis (Brock), hepatitis C, hepatocellular carcinoma,hepcidin deficiency, hereditary hemochromatosis, HIV or other viralillnesses, Huntington's disease, hyperferritinemia, hypochromicmicrocytic anemia, hypoferremia, insulin resistance, iron deficiencyanemia, iron deficiency disorders, iron overload disorders,iron-deficiency conditions with hepcidin excess, juvenilehemochromatosis (HFE2), multiple sclerosis, mutation in transferrinreceptor 2, HFE, hemojuvelin, ferroportin, TMPRSS6 (IRIDA), or othergenes of iron metabolism, neonatal hemochromatosis, neurodegenerativediseases related to iron, osteopenia, osteoporosis pancreatitis,Pantothenate kinase-associated neurodegeneration, Parkinson's disease,pellagra, pica, porphyria, porphyria cutanea tarda, pseudoencephalitis,pulmonary hemosiderosis, red blood cell disorders, rheumatoid arthritis,sepsis, sideroblastic anemia, systemic lupus erythematosus, thalassemia,thalassemia intermedia, transfusional iron overload, tumors, vasculitis,vitamin B6 deficiency, vitamin B12 deficiency, and/or Wilson's disease.

As used herein, “treatment” or “treat” refers to both prophylactictreatment of a subject at risk of, or having a predisposition toward, adisease or disorder, and to therapeutic treatment of a subject sufferingfrom a disease or disorder.

Administration of a therapeutic agent in a prophylactic method can occurprior to the manifestation of symptoms of an undesired disease ordisorder, such that the disease or disorder is prevented or,alternatively, delayed in its progression. Thus, when used inconjunction with prophylactic methods, the term “therapeuticallyeffective” means that, after treatment, a fewer number of subjects (onaverage) develop the undesired disease or disorder or progress inseverity of symptoms.

When used in conjunction with therapeutic methods involvingadministration of a therapeutic agent after the subject manifestssymptoms of a disease or disorder, the term “therapeutically effective”means that, after treatment, one or more signs or symptoms of thedisease or disorder is ameliorated or eliminated.

As used herein, a “hepcidin-related disorder” refers to a conditioncaused by or associated with an abnormal level of hepcidin (e.g.,hepcidin excess or hepcidin deficiency relative to the degree of anemiaor iron stored) which disrupts iron homeostasis. A disruption in ironhomeostasis can in turn result in secondary diseases such as anemia.Acute or chronic inflammatory conditions can result in up-regulation ofhepcidin expression, which can result in decreased circulating ironlevels, which can cause anemia or worsen existing anemia. Exemplaryhepcidin-related inflammatory diseases include anemia of cancer, anemiaof chronic disease, anemia of inflammation, chemotherapy-induced anemia,chronic kidney disease (stage I, II, III, IV or V), end stage renaldisease, chronic renal failure congestive heart failure, cancer,rheumatoid arthritis, systemic lupus erythematosus, Crohn's disease, H.pylori infection or other bacterial infections, hepatitis C, HIV, andother viral illnesses, arteriosclerosis, atherosclerosis, cirrhosis ofthe liver, pancreatitis, sepsis, vasculitis, iron-deficiency,hypochromic microcytic anemia and conditions with hepcidin excess.

As used herein, the phrase “disease (or disorder) of iron homeostasis”refers to a condition in which a subject's iron levels requiremodulation. It includes hepcidin-related disorders; conditions notassociated with elevated levels of hepcidin that nevertheless wouldbenefit from inhibition of hepcidin activity, such as a disruption iniron homeostasis not caused by hepcidin; diseases where aberrant ironabsorption, recycling, metabolism or excretion causes a disruption innormal iron blood levels or tissue distribution; diseases where irondysregulation is a consequence of another disease or condition, such asinflammation, cancer or chemotherapy; diseases or disorders resultingfrom abnormal iron blood levels or tissue distribution; and diseases ordisorders that can be treated by modulating iron levels or distribution.Non-limiting examples of such diseases or disorders of iron homeostasis,hepcidin-related disorders and inflammatory conditions which can resultin hepcidin excess include African iron overload, iron refractory irondeficiency anemia (IRIDA), alpha thalassemia, Alzheimer's disease,anemia, anemia of cancer, anemia of chronic disease, anemia ofinflammation, arteriosclerosis or atherosclerosis (including coronaryartery disease, cerebrovascular disease or peripheral occlusive arterialdisease), ataxias, ataxias related to iron, atransferrinemia, cancer,ceruloplasmin deficiency, chemotherapy-induced anemia, chronicrenal/kidney disease (stage I, II, III, IV or V), including end stagerenal disease or chronic renal/kidney failure, acute kidney injury(AKI), cardiopulmonary bypass-associated AKI, drug or toxin-associatedAKI, cirrhosis of liver, classic hemochromatosis, collagen-inducedarthritis (CIA), conditions with hepcidin excess (elevated hepcidin),congenital dyserythropoietic anemia, congestive heart failure, Crohn'sdisease, Celiac disease, inflammatory bowel disease (IBD), diabetes,disorders of iron biodistribution, disorders of iron homeostasis,disorders of iron metabolism, ferroportin disease, ferroportin mutationhemochromatosis, folate deficiency, Friedrich's ataxia, funicularmyelosis, Gracile syndrome, H. pylori infection or other bacterialinfections, Hallervordan Spatz disease, hereditary hemochromatosis,acquired hemochromatosis, hemochromatosis resulting from mutations intransferrin receptor 2, hemoglobinopathies, hepatitis, hepatitis(Brock), hepatitis C, hepatocellular carcinoma, HIV or other viralillnesses, Huntington's disease, hyperferritinemia, hypochromicmicrocytic anemia, hypoferremia, insulin resistance, iron deficiencyanemia, iron deficiency disorders, iron overload disorders,iron-deficiency conditions with hepcidin excess, juvenilehemochromatosis (HFE2), multiple sclerosis, mutation in transferrinreceptor 2, HFE, hemojuvelin, ferroportin or other genes of ironmetabolism, neonatal hemochromatosis, neurodegenerative diseases relatedto iron, osteopenia, osteoporosis pancreatitis, Pantothenatekinase-associated neurodegeneration, Parkinson's disease, pellagra,pica, porphyria, porphyria cutanea tarda, pseudoencephalitis, pulmonaryhemosiderosis, red blood cell disorders, rheumatoid arthritis, sepsis,sideroblastic anemia, systemic lupus erythematosus, thalassemia,thalassemia intermedia, transfusional iron overload, tumors, vasculitis,vitamin B6 deficiency, vitamin B12 deficiency, and/or Wilson's disease.

Non-inflammatory conditions which are implicated in a disruption of ironregulation include, but are not limited to, vitamin B6 deficiency,vitamin B12 deficiency, folate deficiency, pellagra, funicular myelosis,pseudoencephalitis, Parkinson's disease (Fasano et al., J. Neurochem.96:909 (2006) and Kaur et al., Ageing Res. Rev., 3:327 (2004)),Alzheimer's disease, coronary heart disease, osteopenia and osteoporosis(Guggenbuhl et al., Osteoporos. Int. 16:1809 (2005)), hemoglobinopathiesand disorders of red cell metabolism (Papanikolaou et al., Blood105:4103 (2005)), and peripheral occlusive arterial disease.

In one aspect, provided herein is a method of treating a disorder ofiron homeostasis in a subject in need thereof, comprising administeringto said subject a composition described herein. In another aspect,provided herein is a method of modulating hepcidin activity in a subjectin need thereof, comprising administering to said subject a compositiondescribed herein. In yet another aspect, provided herein is a method fortreating a disorder of iron homeostasis in a subject in need thereof,comprising administering to said subject a composition described herein.In yet another aspect, provided herein is a method of treatinghemochromatosis in a subject in need thereof, comprising administeringto said subject a composition described herein. In yet another aspect,provided herein is a method of treating a subject with an elevated levelof hepcidin, comprising administering to said subject a pharmaceuticalcomposition described herein. In yet another aspect, provided herein isa method of treating anemia in a subject in need thereof, comprisingadministering to said subject a composition described herein. In yetanother aspect, provided herein is a method of treating an inflammatorydisease in a subject in need thereof, comprising administering to saidsubject a composition described herein. In yet another aspect, providedherein is a method of treating an infection in a subject in needthereof, comprising administering to said subject a compositiondescribed herein. An infection may be, for example, a bacterial, fungal,or viral infection.

Any of such methods may, in some instances, further compriseadministering to said subject an erythropoiesis stimulator, wherein saiderythropoiesis stimulator is selected from the group consisting oferythropoietin, an erythropoietin variant and an antibody that bindserythropoietin. In one embodiment, the antibody, or antigen-bindingfragment thereof, that specifically binds hepcidin and saiderythropoiesis stimulator are administered concurrently or sequentially.As used herein, the term “erythropoietic activity” means activity tostimulate erythropoiesis as demonstrated in an in vivo assay, forexample, the exhypoxic polycythemic mouse assay. See, e.g., Cotes andBangham, Nature 191:1065 (1961).

In one embodiment, an antibody, or antigen-binding fragment thereof,described herein and an erythropoiesis stimulator may be used to improvetreatment of a patient with anemia. In another embodiment, patients whoare hypo-responsive to, including unresponsive to, erythropoiesisstimulator therapy, such as erythropoietin or analogs thereof (Epoetinalfa, Epoetin beta, darbepoetin alfa), among others, may benefit fromco-treatment with a hepcidin activity antagonist or hepcidin expressioninhibitor. In another embodiment, an antibody, or antigen-bindingfragment thereof, described herein and an erythropoiesis stimulator maybe used to improve treatment of a patient an iron loading disordersecondary to transfusion-dependent iron overload, or have an ironmaldistribution disorder such as Friedreich's ataxia.

As used herein, “erythropoiesis stimulator” refers to a chemicalcompound that directly or indirectly causes activation of theerythropoietin receptor, for example, by binding to and causingdimerization of the receptor or by stimulating endogenous erythropoietinexpression. Erythropoiesis stimulators include erythropoietin andvariants, analogs, or derivatives thereof that bind to and activateerythropoietin receptor; antibodies that bind to erythropoietin receptorand activate the receptor; or peptides that bind to and activateerythropoietin receptor; or small organic chemical compounds, optionallyless than about 1000 Daltons in molecular weight, that bind to andactivate erythropoietin receptor. Erythropoiesis stimulators include,but are not limited to, epoetin alfa, epoetin beta, epoetin delta,epoetin omega, epoetin iota, epoetin zeta, and analogs thereof,pegylated erythropoietin, carbamylated erythropoietin, mimetic peptides(including EMP1/hematide), mimetic antibodies and HIF inhibitors (seeU.S. Patent Publication No. 2005/0020487, the disclosure of which isincorporated by reference in its entirety). Exemplary erythropoiesisstimulators include erythropoietin, darbepoetin, erythropoietin agonistvariants, and peptides or antibodies that bind and activateerythropoietin receptor (and include compounds reported in U.S. PatentApplication Publication Nos. 2003/0215444 and 2006/0040858, thedisclosures of each of which is incorporated herein by reference in itsentirety) as well as erythropoietin molecules or variants or analogsthereof as disclosed in the following patents or patent applications,which are each herein incorporated by reference in its entirety: U.S.Pat. Nos. 4,703,008; 5,441,868; 5,547,933; 5,618,698; 5,621,080;5,756,349; 5,767,078; 5,773,569; 5,955,422; 5,830,851; 5,856,298;5,986,047; 6,030,086; 6,310,078; 6,391,633; 6,583,272; 6,586,398;6,900,292; 6,750,369; 7,030,226; 7,084,245; 7,217,689; PCT publicationnos. WO 91/05867; WO 95/05465; WO 99/66054; WO 00/24893; WO 01/81405; WO00/61637; WO 01/36489; WO 02/014356; WO 02/19963; WO 02/20034; WO02/49673; WO 02/085940; WO 03/029291; WO 2003/055526; WO 2003/084477; WO2003/094858; WO 2004/002417; WO 2004/002424; WO 2004/009627; WO2004/024761; WO 2004/033651; WO 2004/035603; WO 2004/043382; WO2004/101600; WO 2004/101606; WO 2004/101611; WO 2004/106373; WO2004/018667; WO 2005/001025; WO 2005/001136; WO 2005/021579; WO2005/025606; WO 2005/032460; WO 2005/051327; WO 2005/063808; WO2005/063809; WO 2005/070451; WO 2005/081687; WO 2005/084711; WO2005/103076; WO 2005/100403; WO 2005/092369; WO 2006/50959; WO2006/02646; WO 2006/29094; and US publication nos. US 2002/0155998; US2003/0077753; US 2003/0082749; US 2003/0143202; US 2004/0009902; US2004/0071694; US 2004/0091961; US 2004/0143857; US 2004/0157293; US2004/0175379; US 2004/0175824; US 2004/0229318; US 2004/0248815; US2004/0266690; US 2005/0019914; US 2005/0026834; US 2005/0096461; US2005/0107297; US 2005/0107591; US 2005/0124045; US 2005/0124564; US2005/0137329; US 2005/0142642; US 2005/0143292; US 2005/0153879; US2005/0158822; US 2005/0158832; US 2005/0170457; US 2005/0181359; US2005/0181482; US 2005/0192211; US 2005/0202538; US 2005/0227289; US2005/0244409; US 2006/0088906; US 2006/0111279.

In one embodiment, an antibody, or antigen-binding fragment thereof,described herein and an iron chelator to redistribute iron stores in thebody is also contemplated. An iron chelator is an agent capable ofbinding iron and removing it from a tissue or from circulation. Examplesinclude deferoxamine (Desferal®) and deferasirox (Exjade®), anddeferiprone (1,2-dimethyl-3-hydroxypyridin-4-one).

Administration of a composition herein may be by any suitable meansincluding, but not limited to, injection. In one embodiment, injectionmay be, for example, intravenous, subcutaneous, or intramuscularinjection.

Packages, Kits, and Pre-Filled Containers

Also provided herein are kits containing one or more compounds describedabove. The kit may comprise an antibody or antigen-binding fragmentthereof that binds hepcidin in suitable container means.

The container means of the kits will generally include at least onevial, test tube, flask, bottle, ampoule, syringe an intravenous (IV) bagand/or other container means, into which the at least one polypeptidecan be placed, and/or preferably, suitably aliquoted. Provided herein isa container means comprising a composition described herein.

The kits may include a means for containing at least one fusion protein,detectable moiety, reporter molecule, and/or any other reagentcontainers in close confinement for commercial sale. Such containers mayinclude injection and/or blow-molded plastic containers into which thedesired vials are retained. Kits can also include printed material foruse of the materials in the kit.

Packages and kits may additionally include a buffering agent, apreservative and/or a stabilizing agent in a pharmaceutical formulation.Each component of the kit can be enclosed within an individual containerand all of the various containers can be within a single package.Invention kits can be designed for cold storage or room temperaturestorage.

Additionally, the preparations can contain stabilizers to increase theshelf-life of the kits and include, for example, bovine serum albumin(BSA). Where the compositions are lyophilized, the kit may containfurther preparations of solutions to reconstitute the lyophilizedpreparations. Acceptable reconstitution solutions are well known in theart and include, for example, pharmaceutically acceptable phosphatebuffered saline (PBS).

Packages and kits can further include one or more components for anassay, such as, for example, an ELISA assay. Samples to be tested inthis application include, for example, blood, plasma, and tissuesections and secretions, urine, lymph, and products thereof. Packagesand kits can further include one or more components for collection of asample (e.g., a syringe, a cup, a swab, etc.).

Packages and kits can further include a label specifying, for example, aproduct description, mode of administration and/or indication oftreatment. Packages provided herein can include any of the compositionsas described herein. disease (e.g., IBD), rheumatoid arthritis,osteoarthritis, a forms of cancer and their metastases.

The term “packaging material” refers to a physical structure housing thecomponents of the kit. The packaging material can maintain thecomponents sterilely and can be made of material commonly used for suchpurposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampules,etc.). The label or packaging insert can include appropriate writteninstructions. Kits, therefore, can additionally include labels orinstructions for using the kit components in any method of theinvention. A kit can include a compound in a pack, or dispenser togetherwith instructions for administering the compound in a method describedherein.

In still further embodiments, a kit may further comprise a containermeans for an erythropoiesis stimulator.

Instructions can include instructions for practicing any of the methodsdescribed herein including treatment methods. Instructions canadditionally include indications of a satisfactory clinical endpoint orany adverse symptoms that may occur, or additional information requiredby regulatory agencies such as the Food and Drug Administration for useon a human subject.

The instructions may be on “printed matter,” e.g., on paper or cardboardwithin or affixed to the kit, or on a label affixed to the kit orpackaging material, or attached to a vial or tube containing a componentof the kit. Instructions may additionally be included on a computerreadable medium, such as a flash/cloud drive, disk (floppy diskette orhard disk), optical CD such as CD- or DVD-ROM/RAM, magnetic tape,electrical storage media such as RAM and ROM, IC tip and hybrids ofthese such as magnetic/optical storage media.

Provided herein is a container means comprising a composition describedherein. The container means may be any suitable container which mayhouse a liquid or lyophilized composition including, but not limited to,a vial, syringe, bottle, an in intravenous (IV) bag or ampoule. Asyringe may be able to hold any volume of liquid suitable for injectioninto a subject including, but not limited to, 0.5 cc, 1 cc, 2 cc, 5 cc,10 cc or more.

Provided herein are kits comprising a composition described herein. Inone aspect, provided herein is a kit for treating a disorder associatedwith elevated hepcidin levels or a disorder of iron homeostasis,comprising an antibody, or an antigen-binding fragment thereof, asdescribed herein and an erythropoiesis stimulator.

In another aspect, provided herein is a kit for treating a disorderassociated with elevated hepcidin levels or a disorder of ironhomeostasis, comprising an antibody, or an antigen-binding fragmentthereof, as described herein, and a label attached to or packaged withthe container, the label describing use of the antibody, or anantigen-binding fragment thereof, with an erythropoiesis stimulator.

In another aspect, provided herein is a kit for treating a disorderassociated with elevated hepcidin levels, comprising an erythropoiesisstimulator and a label attached to or packaged with the container, thelabel describing use of the erythropoiesis stimulator with an antibody,or an antigen-binding fragment thereof, as described herein.

EXAMPLES

The application may be better understood by reference to the followingnon-limiting examples, which are provided as exemplary embodiments ofthe application. The following examples are presented in order to morefully illustrate embodiments and should in no way be construed, however,as limiting the broad scope of the application.

Example 1. Monoclonal Antibody Development and Antigen Design to HumanHepcidin

Background

Hepcidin is a 25 amino acid peptide hormone that regulates ironhomeostasis. Genetic or acquired hepcidin deficiency or excess is themain or contributing cause of major diseases of iron regulation. Inother diseases where iron homeostasis is disturbed by the primarydeficiency or excess of body iron stores, blood hepcidin concentrationsreflect the physiologic responses to the primary disturbance. Despitethe potential importance of hepcidin-25 directed therapies in clinicalmedicine, only one humanized monoclonal antibody has entered into earlyPhase 1 clinical studies.

The amino acid sequence of mature hepcidins is highly conserved amongmammals, particularly the N-terminus. Mouse and rat hepcidins are 76%and 64% identical to human hepcidin respectively. The distinctivestructure of hepcidin is highly conserved evolutionarily, and theN-terminal 5 amino acids are absolutely required for bioactivity invitro as the N-terminal amino acids are directly involved in binding Fpnand leading to internalization and degradation in lysozomes (Nemeth etal., 2006).

On the molecular level, this occurs by hepcidin causing the degradationof its receptor, ferroportin, the sole human iron channel, and trappingiron inside liver cells, macrophages, and cells lining the intestinewhere ferroportin is expressed. As plasma iron levels decrease, hepcidinlevels decrease, and ferroportin is produced and trafficked to the cellmembrane. This allows normal iron absorption and recycling to occur andprovide iron required for blood production. The hepcidin-ferroportiniron-regulatory axis is the target of several novel therapeutic drugdevelopment efforts currently in pre-clinical and Phase I trials.However, only one humanized MAb directed against hepcidin is currentlybeing evaluated in early Phase I studies.

Human embryonic kidney (HEK 293) cells have been stably transfected witha ponesterone inducible promoter that promotes high levels of expressionof a murine ferroportin-GFP fusion protein (designated Ec:R50-GFP) tostudy ferroportin biology in vitro cell based assay.

The well-known difficulty in producing antibodies to hepcidin-25 is dueto its compact shape and high degree of evolutionary conservation,particularly in the N-terminal region of the peptide in mammals,including those in use for MAb development efforts.

Other companies have directed their antibodies against linear peptidesfrom the mature C-terminal region of hepcidin-25 (hepcidin (10-25)) [SEQID NO: 27], which is not useful for ELISA detection of biologicallyactive hepcidin-25 and would not be expected to be effective intherapeutic applications (see, e.g., Geacintov et al., US2004/0096990A1).

Antibodies Developed by the Present Inventors

Using our knowledge of the importance of the amino terminus ofhepcidin-25 and its poor immunogenicity, we designed a suite of antigensand immunized groups of BALB/c mice to produce monoclonal antibodies tobiologically active hepcidin-25.

Our antigen design was focused on producing MAbs to the N-terminus ofhepcidin-25 and the full length, oxidized hepcidin-25 peptide. Examplesof antigens that were designed leading to three functional MAbsdescribed herein are shown in FIGS. 1 and 6.

A number of peptide antigens were tested in vivo in BALB/c mice forimmunogenicity and to produce MAbs specific to hepcidin-25 using anumber of synthetic methods to add haptens (e.g., DNP, PamCys) andimmunogenic carrier proteins (e.g., KLH, mKLH, albumin, or CarrierProtein) and few proved to be suitable antigens (FIGS. 5 and 6).

Example 2: Hybridoma Protocol

Following antibody titer calculation, hybridomas are prepared using acommercially available fusion kit (Hy-Clone) or by the conventionalmethods described by Kohler and Milstein (Id.).

The purpose of this example is to describe the production of monoclonalantibodies by isolation of mouse lymphocytes from lymph nodes (LN)and/or spleen (S) after immunization, the production of hybridoma cells,and production and selection of positive clones which secrete theantigen-specific antibodies.

Procedure

Preparation of Myeloma Cells.

Two weeks before fusion, a Sp2/0 myeloma cell line is propagated in DEMEmedium, with 10% FCS, 8-Azaguanine and Pen-Strep. One week prior to cellfusion, the cells are cultured without 8-azaguanine and split the cellsevery other day. Cell density for fusion is 2×10⁵/ml and 100 ml of thesecells are required. The cells were split the day before the fusion andcell viability determined; viability is expected to be greater than 95%.

The SP2/0 cells were harvested by centrifugation at 300×g for 10 minutesand washed 3 times by adding 30 ml of ClonaCell-HY-Fusion Medium B. Thecell pellets were resuspended in 25 ml of Medium B to contain 2×10⁷viable cells. After resuspension the cells were kept at room temperature(RT). This step may be performed simultaneously with, or after, thelymphocyte preparation.

Isolation of Mouse Lymph Nodes, Spleen and Lymphocytes

PEG and media (Medium A, B, C,) were prepared for fusion by pre-warmingto 37° C.

Only mice that responded to immunization as determined by standard serumtiter analysis using hepcidin-25 as the antigen are selected forhybridoma fusion. Hybridoma fusion was performed 3 days after the lastboost with the selected antigen.

Each mouse was sacrificed using asphyxiation and cervical dislocationand sprayed with 75% ethanol. The mouse was placed with its ventralsurface facing up on a dissection board and all limbs secured to thedissection board. All dissection techniques were performed using aseptictechniques and different sets of sterile instruments (scissors, forceps)were used for each step of the dissection to remove the spleen and lymphnodes (LN).

The LN (and/or spleen) was placed into one well of 6 well platecontaining 2 ml of Medium A and the fat and connective tissue trimmedoff. We set one disposable cell strainer on the top of a 50 ml conicalcentrifuge tube and transferred the LN (or spleen) into the strainer andcut LN (or spleen) into small pieces with sterile scissors and groundthe tissue using the plunger of a 3 mL sterile syringe and passing 5-10ml of Medium B through the strainer. We ground the tissue one more timeand rinsed with 10 ml of Medium B (only the membrane should remain onthe screen). The lymphocytes were gently pipetted and mixed by inversionand centrifuged at 400×g for 7 minutes. The supernatant was discared andthe cells re-suspended in 10 ml Medium B. Appropriate dilutions of thecells were then counted using a hemocytometer.

Fusion

The lymphocytes and myeloma cells were mixed at a 4:1 ratio(approximately 8×10⁷ lymphocytes with 2×10⁷ myeloma cells) in a 50 mltube. Lymphocytes and myeloma cells ratio can be in the range from 10:1to 1:1. The fused cells were centrifuged for 10 minutes at 400×g onceand the cell mixture re-suspended in 30 ml Medium B and centrifuged at800 g×5 min to get good adherence and promote fusion of the cells.

The media from was completely aspirated from the cell pellet and thebottom of the tube gently tapped since the pellet must be disrupted foroptimal fusion. One ml of PEG solution was slowly added to the pelletdrop wise using a one ml pipette over a period of one minute withoutstirring. The bottom of the tube was continually tapped gently over thenext minute.

4 ml Medium B was slowly added to the fusion mixture with continuoustapping as before over a period of 4 minutes.

10 ml Medium B was slowly added to the cells, incubate for 15 minutes inwater bath set at 37° C.

30 ml of Medium A was slowly added and the cells centrifuged at 400×gfor 7 minutes. The supernatant was discarded and cells washed with 40 mlof Medium A to ensure that all PEG was removed.

The cell pellet was slowly re-suspended in 10 ml of Clonacell-HYHybridoma Recovery Medium (Medium C) and transferred to a T-75 cm²tissue culture flask containing 20 ml of Medium C (total volume=30 ml).The cells incubated overnight in a humidified incubator at 37° C. in 5%CO₂ atmosphere.

Selection and Cloning

On the day before the fusion, the ClonaCell-Hy Hybridoma SelectionMedium (Medium D) was placed at 2-8° C. and thawed overnight. On the dayof the fusion, Medium D was shaken vigorously to mix contents well andlet warm to room temperature.

The fused cell suspension was then transferred into a 50 ml conical tubeand centrifuged for 10 minutes at 400×g at RT and the supernatantremoved and discarded. The cells were re-suspended in Medium C to atotal volume of 10 mL.

10 mL of the cell suspension was transferred into 90 mL of Medium D andmixed thoroughly by gently inverting the bottle several times. Thehybridoma cell suspension was then transferred into a disposable reagentreservoir and allowed to sit for 15 minutes at RT to let any bubbles torise to the top and disperse.

Using a multi-channel pipette and sterile pipette tips, theClonaCell®-HY Medium D containing the hybridoma cells was dispensed in60-80 μL volumes per well into 96-well plates. This typically yieldedbetween 10-16 plates depending on the volume plated. The plates wereincubated at 37° C. in a humidified, 5% CO₂ incubator. Following 8 daysof undisturbed incubation, the wells were examined for the presence ofcolonies and gently overlain with 150 μL of pre-warmed ClonaCell®-HYMedium E onto the semi-solid medium of each well, regardless of thepresence of colonies and analysis performed on all wells.

The plates were incubated for an additional 2-4 days at 37° C. in ahumidified, 5% CO₂ incubator. The overlay incubation time may beincreased further to ensure the detection of low expressing hybridomas.

100 μL of the overlaid ClonaCell®-HY Medium E was carefully removedwithout disturbing the colonies in the semi-solid medium. Thesupernatants were tested for specific antibodies using an assay systemappropriate for the antigen involved e.g. Neutravidin ELISA, Mouse MabIsotyping etc.

The contents of wells that tested positive for antibodies were gentlyre-suspended and transferred to wells of a 24-well plate containing onemL of ClonaCell®-HY Medium E to expand the hybridomas. When a positivewell contained more than a single colony the clones were harvestedseparately and transferred to individual wells for expansion andretesting to determine which clone produces the antibody of interest.

Freezing Hybridomas

Cells were cryopreserved at a concentration of 2×10⁶ cells per vial.

A 20% DMSO solution in Fetal Bovine Serum (FBS) was placed in a 50 mLconical tube and allowed to cool on ice. The appropriate volume of DMSOwas slowly added and mixed well and filter sterilized using a 0.2 μmfilter and keep on ice.

Harvest cells and re-suspend in cold FBS at twice the desired final cellconcentration (e.g., suspend at 4×10⁶ cells/mL for cells cryopreservedat 2×10⁶ cells per cryovial).

For cryopreservation, the FBS/20% DMSO solution was slowly added at aratio of 1:1 to the tube containing the cells with continuous mixingduring the addition. One mL of cells in freezing medium was transferredto each cryovial. The final cell suspension was calculated be in 90% FBScontaining 10% DMSO.

Cryovials were placed immediately into freezing containers and thenmoved into −80° C. freezer overnight. Next day, remove frozen vials fromthe freezing container and store in liquid nitrogen.

Example 3: Screening Hybridomas for Anti-Hepcidin Antibodies

After eight days of undisturbed incubation following fusion, all wellswere screened to identify murine hybridomas that secreted anti-humanhepcidin antibodies. Briefly, one day before screening 100 μl ofneutravidin (200 ng/well) prepared in carbonate coating buffer (pH 9.6)was placed into each well of an enzyme immunoassay (EIA) plate andincubated overnight at 4° C. The following day the plate was washed,blocked with 1% BSA in buffer, and 1 ng of K18-Biotin hepcidin-25 tracerwas added to each well. After one hour incubation, the plate was washed,100 μl of hybridoma tissue culture supernatant was combined with 50 μlof 1% BSA/TBST in each well and the plate incubated on a rotary shaker(240 rpm) for 1.5 h. The plate was washed and a HRP-labeled goatanti-mouse IgG (H+L) chain detection antibody was added and incubationcontinued for another hour. The plate was washed, substrate applied, thereaction developed for 10 min before stopping with 1N H₂SO₄ and theabsorbance read at 450 nM. An example of this first round screen yieldedan 8×12 matrix of OD values as depicted in FIG. 2. Hybridomas thatproduced an OD >2.0 were identified and further propagated prior to thesecond round screen.

The second round of screening involved testing the specificity of eachhybridoma to hepcidin-25. Briefly, 96 well EIA plates are coatedovernight with goat anti-mouse IgG Fc-specific antibody (1/2,500dilution), and the following day the plate was washed, blocked and 100μl of tissue culture supernatant was placed in each well and incubatedfor 1 hour at room temperature. The ability of the murine antibodiespresent in the tissue culture supernatant that were captured by the Fcregion was tested by addition of 1 ng of K18-Biotin Hepcidin-25 tracerto each well and incubating for 1.5 hours on a rotary plate shaker.After washing, HRP labeled streptavidin (SA-HRP) was added to the wells,incubated for one hour, washed and the presence of antibody-capturedtracer was detected by the addition of TMB substrate for 10 min. Thereaction was halted by the addition of acid and absorbance read at 450nM. As depicted in FIG. 3, hybridomas that produce an OD>0.4 wereidentified and subcloned for further characterization. Our experiencehas proven that hybridomas that produce an OD that exceeds 2.0 in thissecond round of screening are strong candidates for furthercharacterization.

To test for functional activity of the hybridomas, clones identified inthe second round screen were subcloned, grown to approximately 70%confluency and screened as described for the second round screen.Briefly 96-well plates are coated with goat anti-mouse IgG Fc-specificantibody and the following day the plates were washed and blocked andtissue culture supernatant was placed in each well and incubated for 1hour.

To prepare a stock solution of hepcidin, weigh out approximately 1 mg oflyophilized Hepcidin and reconstitute in 0.5 ml of 0.016% HCl (preparedin sterile tissue culture grade water) to make a final concentration of2 mg/ml.

To accurately determine hepcidin concentration in the solution, measurethe absorbance at 215 nm and 225 nm on a spectrophotometer.

For sample measurement, make a 1:20 dilution of the solution (10 μl ofthe stock solution to 190 μl of sterile water). Blank thespectrophotometer with 10% of 0.016% HCl. (10 μl of the 0.016% HCl to190 μl of sterile water). Measure the absorbance at 215 nm and 225 nmand calculate the hepcidin concentration using the following formula forpeptide concentration:

The calculation for hepcidin-25 concentration in mg/ml is [hepcidin-25mg/ml]=(A215-A225)×0.144×20 (20 is the dilution factor).

To store hepcidin-25 stock solutions aliquot the solution in 100 μlvolumes into a sterile 0.5 ml microcentrifuge tubes and store at −80° C.To further dilute the hepcidin stock to a convenient working solution,dilute the concentrated stock to 500 μg/ml (working solution) withsterile tissue culture grade water.

To confirm peptide concentration in the prepared solution, measure theabsorbance at 215 nm and 225 nm. For the measurement, make a 1:10dilution of the solution. Aliquot in 25 μl volumes in 0.5 ml sterilemicrocentrifuge tubes with the screw cap.

The ability of synthetic human hepcidin-25 to compete against theK18-Biotin Hepcidin-25 tracer for MAb binding sites was tested by theaddition of 100 ng of hepcidin-25 prepared as above to the tracersolution and 100 μl of this was added to each well and incubated for 1.5h. After washing, HRP labeled streptavidin was added, incubated for onehour, washed and the presence of bound tracer was detected by theaddition of TMB substrate. The reaction was halted by the addition ofacid and absorbance read at 450 nM. An example of functional activityscreening of hybridoma supernatants is shown in FIG. 4. Hybridomas thatdisplay functional activity were identified by a reduction in the OD inthe Tracer+Hepcidin-25 wells, compared to the OD produced by the Traceronly (for example hybridoma 5A3; 5A3 designation was later changed to“MAb 583”).

An example of the difficulty of producing murine MAbs is shown in FIG.5. A variety of typical antigens and immunization approaches can yieldvarying numbers of mice that respond to immunization (based on serumtiters), and the tissues that yield successful fusions. Regardless ofthe antigen employed, the percentage of functional hepcidin-25 specificmurine MAbs is consistently less than 0.06% (FIG. 5). Similarly, as anexample of the effort required for discovery of 3 murine MAbs specificfor hepcidin-25, we tested eight antigens and screened 11,845 hybridomaswith a success rate of 0.025% (FIG. 6).

Example 4: Purification of Murine MAbs

Large quantities of MAb 583 and MAb 1B1 were produced by seedingindividual commercially available CellMax hollow fiber bioreactors(10,000 cm² surface area (Spectrum Labs, Inc.) that were then incubatedat 37° C. in a 5% CO₂ atmosphere. Approximately 2-3 liters of total cellculture supernatant from each MAb was harvested in approximately 100 mlbatches. Each batch was centrifuged and frozen at −20° C. untilpurification.

For purification, the supernatant was thawed, re-centrifuged andimmunoglobulin was purified by affinity chromatography using a 5 mlHiTrap protein G column (GE Healthcare, Uppsala, Sweden) as permanufacturer's instructions. Purification was performed using a BioRadBiologic DuoFlow medium pressure chromatography system equipped with aBioLogic Maximizer, QuadTec UV-VIS detector and a BioFrac fractioncollector. For SDS-PAGE analysis. Flow through fractions from twoprevious purifications of MAb 583 and MAb 1B1 containing immunoglobulinwere pooled and buffer exchange was performed using a HiPrep 26/10desalting column (GE Healthcare). Protein concentration was determinedusing bicinchoninic acid (BCA, Thermo Scientific) and aliquots werestored frozen at −20° C.

An example of the purification of MAb 581 and MAb 1B1 (lot 3) wasconfirmed using Coomassie stained SDS-PAGE gels (12% acrylamide) rununder standard reducing conditions (FIG. 7). As depicted in FIG. 7,purified preparations of MAbs 583 and 1B1 IgG (lanes 8, 9) were obtainedfrom their respective hybridoma culture supernatants (lanes 2-5) asevidenced by the presence of both heavy and light chain proteins of thecorrect molecular mass (approximately 50 and 25 kDa, respectively). Inaddition, the purified murine MAbs were electrophoretically equivalentto the mouse IgG control (lane 10). Serum free tissue culture medium(lane 6) served as a negative control. This method would consistentlyyield a purified MAb that was suitable for further binding affinity(e.g. Biacore) and specificity studies.

To assess consistency of bioreactor production and purification yield aMAb activity characterization method was developed to assess the bindingactivity each successive purification Lot of MAb 583 antibody that wereeach assessed by gel electrophoresis as shown in FIG. 7. This samemethod was applied to purifications of MAb 1B1 (data not shown).

We used a microwell plate ELISA to assess antibody activity. Serialdilutions of purified MAb 583 from each successive purification Lot frombioreactor supernatants (approx. 100 ml per Lot) were coated on plates(0-100 ng) and blocked. The NT-biotin hepcidin-25 tracer was addedacross the plate at 1 ng/well in TBST, pH 8) containing 0.25% Blotto.The tracer was allowed to bind for 2 hours and the wells were washed inTBST, pH 8 with no Blotto. SA-HRP was added at 1:2500 for 30 minutes,the wells washed in TBST, and TMB substrate added for 10 minutes. Stopbuffer was added the OD quantified on a spectrophotometer at 450 nm. AnOD of 4 indicates the absorbance is beyond the analytic range of thespectrophotomer.

The table below is an example of the established ELISA method todetermine binding activity characteristics for 5 successive MAb 583purifications.

Lot 3 Lot 4 Lot 5 Lot 6 Lot 7 583 (ng/well) 583 #3 583 #4 583 #5 583 #6583 #7  100 ng/well 4 4 4 4 4  50 ng/well 4 4 4 4 4  25 ng/well 4 4 4 44 12.5 ng/well 4 4 4 4 4 6.25 ng/well 2.1401 3.2808 2.7839 4 3.3373 3.12ng/well 1.0645 1.8302 1.5279 2.3104 2.1237 1.56 ng/well 0.5974 1.15270.9335 1.4203 1.3805   0 ng/well 0.0446 0.0257 0.0232 0.0303 0.0642

The data show that the binding activity of purified MAb 583 across lotsincreased after Lot 3 and was consistent across Lots 4-7 after thepurification protocol was optimized over the first four purificationsperformed. Wells coated with 6.25 ng purified MAb 583 demonstrated thatoptical density at 450 nm ranged from 2.7-4 across Lots 4-7 and at thenext dilution (3.12 ng/well) the ODs ranged from 1.5-2.3. This exampledemonstrates that an established and validated protocol for purificationof the MAb 583 antibody from bioreactor supernatants is established andshown to be consistent over dozens of purification Lots of both MAb 583and 1B1.

Example 5: Characterization of MAb Specificity to Hepcidin-25 andHepcidin Peptides

MAb 583 demonstrates exquisite and excellent specificity for hepcidin-25in a solution based specificity assay. For example, hepcidin-25 wascoated on a maleic anhydride plate and unoccupied binding sites werequenched using standard methods. In parallel, 20 ng of MAb 583 is mixedwith increasing concentrations of hepcidin-25 (0 to 8 ng/well) in a lowprotein binding 96-well plate and allowed to react for one hour. ThisMAb mixture is then transferred to the maleic anhydride plate andallowed to react for 2 hours, after which it is washed and the presenceof MAb 583 bound to hepcidin-25 covalently bound to the plate isdetermined using HRP-goat anti-mouse IgG secondary antibody. Afteradditional incubation and washing the substrate is added, developmenthalted using acid and the OD 450 nM is determined. FIG. 8 demonstratesthat hepcidin-25 can block the binding sites on MAb 583 in solution in adose dependent manner.

Another example of the specificity of MAb 583 for hepcidin-20,hepcidin-22 and hepcidin-25 and protegrin was demonstrated usingmembrane based assays such as, but not limited to, non-reducing tricineSDS-PAGE (10-20% acrylamide) analysis using standard electrophoresis andimmunoblot conditions (FIG. 9). As depicted in the Coomassie stainedimage, hepcidin-20, -22 and -25 (lane 2, 3, 4) and protegrin (lane 5)all have a similar molecular mass of approx. 3 kDa. In contrast, theWestern blot probed with MAb 583 indicated that MAb 583 specificallyrecognized hepcidin-20, hepcidin-22 and hepcidin-25 but did notrecognize protegrin.

To further exemplify the specificity of MAb 583 and MAb 1B1 forhepcidin-22 and hepcidin-25 and NT-biotin hepcidin-25 tracer, K18-biotinhepcidin-25 tracer and K24-biotin hepcidin-25 tracer, reducing SDS-PAGE(12% acrylamide) analysis and Western immunoblots were performed understandard electrophoresis and immunoblotting methods (FIG. 10). Coomassiestained SDS-PAGE analysis indicates that both hepcidin-20 andhepcidin-25 (lane 2, 3) and the three forms of biotin-labeled tracer(lane 4, 5, 6) have an identical molecular mass. Western blots probedwith either MAb 583 or MAb 1B1 indicated that these MAbs specificallyrecognized the hepcidin-20, hepcidin-25 and the three forms ofbiotin-labeled hepcidin-25 tracer peptides. Taken collectively, oursolution-based and membrane based studies demonstrate that MAb 583 andMAb 1B1 possess unique specificity for all three forms of thebiotinylated human hepcidin-25 tracer molecules.

Example 6: BIAcore Surface Plasmon Resonance (SPR) Analysis of MAbs 583and 1B1

This example describes the analysis of the binding affinity anddissociation constants of MAbs 583 and 1B1 interaction with hepcidin-25using SPR.

SPR was performed on a Biacore 3000 System (BIAcore, Piscataway, N.J.)using CMS sensor chip. CMS chip matrix consists of a carboxymethylateddextran covalently attached to a gold surface. All measurements wereperformed at 25° C.

Neutravidin (Sigma, St. Louis, Mo.) was immobilized on a CMS sensor chip(flow cells 1 to 4) by the amine-coupling protocol, at a level of5000-10000 response units (RUs). The amine coupling protocol includesactivation of the dextran matrix on the sensor chip surface with a 1:1mixture of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl carbodiimide (EDC) and0.1 M N-hydroxysuccinimide (NHS), followed by injection of neutravidinin 10 mM sodium acetate buffer, pH 4.

After neutravidin immobilization, the subsequent steps were carried outin HBS-EP buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, and0.005% surfactant P20).

Biotinylated hepcidin peptides (NT-biotin-hepcidin-25,K18-biotin-hepcidin-25 and K24-biotin-hepcidin-25) were immobilized inflow cells 2-4 (one peptide species per flow cell) by injectingindividual peptides at a concentration of 200 μg/ml at a flow rate of 5μl/min, for 20 min.

After biotin-hepcidin-25 analogs were captured on the chip,anti-hepcidin MAb 583 or 1B1 was injected into flow cells 1-4 at theconcentration of 24 μg/ml in HBS-EP buffer at the flow rate of 50μl/min, for 3 min. After 3 minutes, injection was stopped anddissociation was followed for 6 min. Regeneration was performed byinjecting 10 mM glycine HCl, pH 1.5 at a flow rate of 10 μl/min for 1min.

Resonance signals were corrected for nonspecific binding by subtractingthe signal of the control flow cell (cell 1) and analyzed usingBIAevaluation 4.1 software (Biacore).

In the first SPR experiment, MAb 583 was applied to the Biacore chipprepared as described above at a concentration of 24 μg/ml and rapidbinding and very low rate of dissociation of MAb 583 to the threebiotin-hepcidin-25 analogs was observed (FIG. 11). The data from the SPRexperiments shown in FIG. 11 are shown in the table below. Excellentbinding affinity (Ka) and dissociation constants (Kd) were observed forMAb 583 with the NT-biotin hepcidin-25, and approximately 1 log loweraffinity and dissociation constants for K18-biotin hepcidin-25 andK24-biotin hepcidin-25, respectively.

ka kd Rmax RI Conc of KA KD Req kobs (1/Ms) (1/s) (RU) (RU) analyte(1/M) (M) (RU) (1/s) 24 μg/ml Ab583 to 6.11e4 3.15e−6 78.8 18.7 160n1.94e10 5.16e−11 78.8 9.77e−3 NT-biotin-hepcidin-25 24 μg/ml Ab583 to6.37e4 5.59e−5 127 25.9 160n 1.14e9  8.78e−10 126 0.0102K18-biotin-hepcidin-25 24 μg/ml Ab583 to  5.2e4 2.88e−4 65.3 2.11 160n1.8e8 5.54e−9  63.1  8.6e−3 K24-biotin-hepcidin-25

We repeated the Biacore experiment shown in FIG. 11 with anapproximately 5-fold lower MAb 583 concentration (5 μg/ml) to assess MAb583 at a much lower molar ratio of antibody for the two bestbiotinylated hepcidin-25 antigens, NT-biotin hepcidin-25 and K18-biotinhepcidin-25 (FIG. 12). The Biacore plot shows MAb583 has excellentbinding affinity and very low disassociation from both K18-biotinhepcidin-25 and NT-biotin hepcidin-25 peptides assessed in this Biacoreexperiment. Biacore results from FIG. 12 are shown in FIG. 13.

The Biacore data indicate that MAb 583 binds to K18-biotin hepcidin-25with high affinity and a low picomolar dissociation constant(Kd)=approx. 7.5 pM). MAb 583 has high but slightly lower bindingaffinity and low picomolar dissociation constants forNT-biotin-hepcidin-25, with a Kd=approx. 18 pM.

These Biacore experiments and results confirm that MAb 583 bindshepcidin-25 rapidly and with high affinity and dissociates fromhepcidin-25 slowly with low pM dissociation constants. The epitope forMAb 583 is the N-terminal 9 amino acids of which the first 5 N-terminalamino acids (SEQ 25) are essential for hepcidin-25 binding to the irontransporter and receptor, ferroportin, and its ability to internalizeand degrade ferroportin The excellent specificity, affinity, avidity,and pM Kd of MAb 583 for the N-terminus of hepcidin-25 indicates that itwill be a neutralizing antibody in vitro and in vivo, and suitable forhumanization for therapeutic applications. Examples of Biacoreexperiments with MAb 1B1 are shown in FIGS. 14-15. Two attempts weremade using the same conditions as described for MAb 583 to conduct SPRanalysis with MAb 1B1 in and each case the dissociation rate of 1B1 fromthe hepcidin-25 antigens was so low that the Biacore 3000 instrumentthat the BIAevaluation 4.1 software used could not detect anydissociation of 1B1 from the hepcidin-25 antigens over the 20 minuteexperiment. For this reason, the experiments failed to producestatistical information as shown for MAb 583 in FIG. 13 for the Biacoreexperiments shown in FIGS. 14-15.

SPR analysis under these experimental conditions indicate that themurine MAb 1B1 has extraordinary affinity for hepcidin-25 peptides andmay have an affinity constant (K_(D)) of ≦10⁻¹²-10⁻¹³ M) and with thesecharacteristics may be suitable for humanization and pre-clinicalevaluation as a hepcidin-25 specific MAb.

Example 7: Hepcidin Specificity and Binding Experiments with MAbs 583and 1B1

The specificity and relative binding affinities of the 583 and 1B1 wereassessed in a series of microtiter plate competition experiments. Assayswere performed in duplicate or triplicate using 96-well-microtiterplates coated with MAb 583 or MAb 1B1.

MAb 583 was diluted 1:4000 in Tris buffered saline (TBS) containing 40mM Tris-HCl (pH 7.3), 100 mM NaCl, was pipetted into the microtiterplates.

After a 1 hour incubation at room temperature (RT), the microtiterplates were washed with TBST (TBS with 0.05% TWEEN® 20) and 100 mlstandard samples containing various amounts of synthetic peptides andbiotin hepcidin-25 analogs (1-2 ng/well) were added to each well andincubated for 1 hour at RT.

Competition was detected by streptavidin-HRP with the substratetetramethylbenzidine; the color reaction was stopped with 0.5 N H₂SO₄and the optical density of the solution read at 450 nm wavelength.

Analysis of binding of hepcidin-25, hepcidin-22, and hepcidin-20 to MAb583 antibodies coated on a microtiter plate. Binding of biotinylatedhepcidin-25 analogs and detection of bound NT-biotin hepcidin-25 wasused to detect relative degree of binding of each of the hepcidinpeptides relative to hepcidin-25 (FIG. 16). Competition curves with theNT-biotin hepcidin-25 with the hepcidin peptide isomers, hepcidin-22 andhepcidin-20, were similar indicating that the hepcidin isomers do bindto MAb 583, but with decreasing EC50 values (affinity) with decreasingsize of the hepcidin isomer as demonstrated by the right shift in theregression curve going from hepcidin-25 to hepcidin-22 to hepcidin-20.

We used the same method as above to investigate the relative bindingaffinities of MAbs 583 and 1B1 against a C-terminal, oxidized peptide,to assess the binding epitopes on hepcidin for each MAb. The carboxyterminal peptides such as hepcidin (10-25) are described in U.S. Pat.Nos. 7,320,894 and 7,411,048, each of which patents are incorporatedherein by reference with respect to the peptides.

As shown in FIG. 17 and FIG. 18, there was no binding of the hepcidin(10-25) peptide to either MAb 583 or 1B1, respectively, at any testedconcentration up to 2000 ng/ml with either the NT-biotin hepcidin-25.There was also no binding observed between MAb 583 and mouse hepcidin-25(murine hepcidin-1) or protegrin as compared to excellent competitivebinding by synthetic hepcidin-25 in this ELISA experiment (FIG. 17).These experiments clearly show that MAbs 583 and 1B1 do not bind anyepitopes found in the C-terminal 16 amino acids of hepcidin-25 (hepcidin(10-15) and thus bind N-terminal epitopes.

The cationic antimicrobial peptide, protegrin, and mouse hepcidin-25,(murine hepcidin-1) are structurally similar, with murine hepcidin-1sharing 76% amino acid identity to human hepcidin-25. We used to thepeptides to test cross-reactivity of MAb 583 with similar peptides inthe same assay with MAb 583. As clearly shown in an ELISA experiment, wefound no apparent binding of 583 to these structurally similar peptides(FIG. 17).

In a similar experiment as shown in FIG. 18, we observed no binding ofhepcidin (10-25) to MAb 1B1 in a similar experiment conducted withK18-biotin hepcidin-25 used as the tracer in the ELISA experiment (FIG.19) further confirming the N-terminal 9 amino acids as the key epitopefor 1B1. Importantly, these data show that both MAbs 583 and 1B1 haveexcellent affinity and specificity for the N-terminus of hepcidin-25.Both would be predicted to be neutralizing antibodies for hepcidin'sbioactivity against the ferroportin receptor and iron channel in vitroand in vivo and once humanized, candidates for therapeutic development

Example 8: Analysis of MAb 583 for Neutralizing Activity AgainstHepcidin-25 In Vitro in Cell Based Assays by Ferroportin-GFPFluorescence Analysis

In Vitro Cell Based Assays

We assessed the neutralizing activity of MAb 583 in vitro in a cellbased fluorescence assay using the flow cytometry protocol as describedin Nemeth et al. (2006) to assess the neutralizing activity of MAb 583against human hepcidin-25 (SEQ ID NO. 19). The N-terminal five aminoacids [SEQ ID NO. 25] of hepcidin interact with ferroportin and arerequired for the biological activity of hepcidin, whereby each singleamino acid deletion from the N-terminus reduces hepcidin's biologicalactivity as defined by ferroportin degradation activity (FIGS. 20-23).

Human HEK cells containing a ponasterone-inducible mouse ferroportinconstruct (Fpn-GFP) were incubated with or without 10 mM ponasterone for24 hours. After three washes with 1× Dulbecco's PBS, the cells weretreated sequentially with known quantities of Protein A affinitypurified MAb 583 antibody and known concentrations of biologicallyactive synthetic human hepcidin-25, or control buffer for another 24hours.

Cells were detached using TrypLE Express (Invitrogen) and re-suspendedin medium at a concentration of 1×10⁶ cells/ml. The intensity of greenfluorescence was measured using flow cytometry.

Cells not expressing Fpn-GFP (no ponasterone) were used to establish agate (baseline) to exclude background fluorescence. The results wererepresented as a fraction of the GFP intensity of untreated cells,according to the formula (Fx−Fhep)/(Funtreated−Fhep), where F representsthe mean of the gated green fluorescence.

Flow Cytometry of Fpn-GFP Cells Treated with MAb 583.

In the first experiment, cells were induced overnight with ponasteroneto induce expression of murine Fpn-GFP. Next day, ponasterone wasremoved by washing, and hepcidin-25 and 583 antibodies added for 24hours (FIG. 20 and FIG. 21).

Hepcidin-25 was used at 100 ng/ml concentration (37 nM). MAb 583 wasadded at 10-times, 2-times or ⅓rd relative molar concentration ofhepcidin concentration (370 nM, 74 nM and 10 nM).

The control MAb was a failed anti-hepcidin monoclonal antibody whenscreened in vitro by ELISA and was used at the highest concentration(370 nM). In this experiment, 10 nM MAb 583 completely neutralized 37 nMhepcidin-25 and suppressed hepcidin-25 degradation of FPN-GFP.

We repeated the experiment by with MAb 583 added at ⅓rd, ⅙th, 1/12th,and 1/24th the molar concentration of hepcidin-25 in these cell basedassay of MAb 583 biological activity (FIG. 22 and FIG. 23).

In this experiment 2.5 nM of MAb 583 neutralized significantly (˜23%decrease) 37 nM hepcidin-25 and it biological activity to FPN-GFP at1/12^(th) of the molar ratio of biologically active hepcidin-25 (FIG.22, 23).

We also assessed the neutralizing activity of MAb 583 by obtainingintracellular ferritin measurements from control Fpn-GFP cells and cellstreated with varying concentrations of MAb 583 and hepcidin in twoadditional experiments using the identical protocols, includingconcentration of the MAb 583 antibody and hepcidin-25 biotinylatedpeptides, as in the cell based fluorescence assays (FIGS. 24-26).

To obtain intracellular ferritin concentrations, total cellular proteinwas extracted using RIPA buffer (Boston BioProducts, Ashland, Mass.)with addition of a protease inhibitor cocktail according to themanufacturer's instructions (Roche, Indianapolis, Ind.).

Ferritin levels were determined using an enzyme-linked immunosorbentassay (ELISA; Ramco Laboratories, Stafford, Tex.) according to themanufacturer's instructions with normalized total protein concentrationsin each sample. Total protein concentration was determined using thebicinchoninic acid (BCA) assay (Pierce, Rockford, Ill.).

FIGS. 24-26 show the results of these assays designed to assess theability of MAb 583 to neutralize the biological activity of hepcidin-25against ferroportin.

Similar to the results observed in fluorescence assays described herein,2.5-10 nM MAb 583 significantly neutralized 37 nM hepcidin-25 and itsbiological activity in vitro, leading to decreased degradation ofFPN-GFP and retention of intracellular ferritin bound iron (FIG. 24-26).

Example 9. In Vivo Neutralizing Activity of MAb 583 in C57BL/6 Mice

To assess the in vivo neutralizing characteristics of MAb 583, weperformed a simple but robust animal study were we examined two dosingregimens with affinity purified MAb 583 antibodies for the 583antibodies ability to block biologically active hepcidin-25 in vivo. Wetested a single dose of MAb 583 and two 50% doses of MAb 583 appliedsequentially 24 hours apart via intra peritoneal injection.

To initiate the in vivo MAb 583 experiment, forty C57BL/6 male mice (6weeks of age) were housed in a commercial vivarium within our buildingand fed a low iron diet (20 ppm total iron, Teklad Custom Research Diet,Harlan Laboratories) for 17 days. On day 1, forty mice were randomizedinto five experimental groups of 8 and each group was treated asdescribed below. Group one received PBS only, groups 2 and 3 received1.0 mg and 0.5 mg of MAb 583 respectively, group 4 received the controlMab (anti-hepcidin MAb unsuitable for ELISA) and group 5 received PBS.Twenty-four hours later each mouse in group 3 received an additional 0.5mg of MAb 583. Following an additional 24 hour incubation period, groups1 through 4 received 50 μg hepcidin-25 in PBS and mice in group 5(control group) received their second dose of PBS (FIG. 27)

Mice were bled via cardiac puncture 2 hours after treatment, blood wasallowed to clot for 30 min and their serum iron levels assessed using acommercial spectrophotometric method (Iron-SL Assay, GenzymeDiagnostics).

Statistical analysis of the data show the data was normally distributedby the Shapiro-Wilk Normality Test (P=0.216).

The Equal Variance Test of the data showed equivalent variances acrossthe groups (P=0.360).

ANOVA was performed and indicated that there was a statisticallysignificant difference (P=0.008) differences in the mean values of serumiron concentrations among the treatment groups (FIG. 28). FIG. 27 showsthese results graphically and the significant differences between PBScontrols and mice treated with either hepcidin-25 in PBS alone or incombination with two 0.5 mg doses of MAb 583 in PBS (FIG. 28).

Power of performed test with alpha=0.050:0.748.

Multiple Comparisons versus Control Group (Holm-Sidak method) yielded anoverall significance level=0.05.

Significant differences were observed in plasma iron concentrationsbetween the PBS control group and the group of mice administered either50 μg hepcidin-25 in PBS or two 0.5 mg doses of MAb 583 over 24 hours.The group administered the 50 μg hepcidin-25 in PBS followed by 1.0 mgof the control anti-hepcidin MAb (sham MAb) approached a significantdifference with the PBS plus hepcidin-25 control group (P=0.054; FIG.28).

These results are promising and indicate that MAb 583 has neutralizingactivity in vivo that suppress the biological activity of hepcidin-25when both are injected sequentially IP in male C57BL/6 mice. The 50 μgdose of human hepcidin-25 used in this in vivo experiment has beenpreviously shown to induce severe hypoferrimia for up to 72 hours inC57BL\6 mice and therefore the dose represents a stringent test of theneutralizing activity of MAb 583 (Rivera et al. 2005).

Example 10. Human-Mouse Chimeric Antibody

The light and heavy chain variable domains of murine anti-hepcidin MAB583 were synthesized and cloned into a proprietary mammalian expressionvector without any modifications. The light chain variable domain wascloned in-frame with a secretion signal and a human kappa light chainconstant domain. The heavy chain variable domain was cloned in framewith a secretion signal and a human IgG1 constant domain. The resultingclone, BAP070-01, was sequence verified. Note that the signal sequenceis underlined below.

BAP070-01-Light Chain DNA Sequence

(SEQ ID NO: 37) ATGGACATGAGGGTCCCCGCTCAGCTCCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCAAGTGTGACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATATCCTGCAGAGCCAGTGAAAGTGTTGATAGTTATGGCAATAGTTTTATGCACTGGTATCAGCAGAAACCAGGACAGCCACCCAAACTCCTCATCTATCGTGCATCCAACCTAGAATCTGGGATCCCTGCCAGGTTCAGTGGCAGTGGGTCTAGGACAGACTTCACCCTCACCATTAATCCTGTGGAGGCTGATGATGTTGCAACCTATTACTGTCAGCAAAGTAATGAGGATCTGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAAC AGGGGAGAGTGT

Amino acid sequence of BAP070-01-Light Chain Protein

(SEQ ID NO: 38) MDMRVPAQLLGLLLLWLPGAKCDIVLTQSPASLAVSLGQRATISCRASESVDSYGNSFMHWYQQKPGQPPKLLIYRASNLESGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQSNEDLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC

BAP070-01-Heavy Chain DNA Sequence

(SEQ ID NO: 39) ATGGCCACAACCATGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTGGCTATTTTAAAAGGTGTCCAGTGTCAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGGTATACCTTCACAAACTATGGAATGAACTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAACACCTACACTGGAGAGCCAACATATGCTGATGACTTCAAGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAACAACCTCAAAAATGAGGACACGGCTACATATTTCTGTACAACGTACGCTACTAGCTGGTACTGGGGCCAGGGAACGCTGGTCACCGTCAGCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCAGCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA

Amino acid sequence of BAP070-01-Heavy Chain Protein

(SEQ ID NO: 40) MATTMEFGLSWLFLVAILKGVQCQIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCTTYATSWYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Murine mAb 583 Heavy Chain Variable Region Sequence:

(SEQ ID NO: 53) CAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGGTATACCTTCACAAACTATGGAATGAACTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAACACCTACACTGGAGAGCCAACATATGCTGATGACTTCAAGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAACAACCTCAAAAATGAGGACACGGCTACATATTTCTGTACAACGTACGCTACTAGCTGGTACTGGGGCCAAGGCACCAC TCTCACAGTCTCCTCA

Murine mAb 583 Light Chain Variable Region Sequence:

(SEQ ID NO: 54) GACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATATCCTGCAGAGCCAGTGAAAGTGTTGATAGTTATGGCAATAGTTTTATGCACTGGTATCAGCAGAAACCAGGACAGCCACCCAAACTCCTCATCTATCGTGCATCCAACCTAGAATCTGGGATCCCTGCCAGGTTCAGTGGCAGTGGGTCTAGGACAGACTTCACCCTCACCATTAATCCTGTGGAGGCTGATGATGTTGCAACCTATTACTGTCAGCAAAGTAATGAGGATCTGACGTTCGGTGGAGGCACCAAGCTGGAAATCAA AC

CHO cells were seeded in 6 well plates, transfected with BAP070-01(recombinant chimera) or empty vector only (BAP070) using a proprietarytransfection protocol and cultured at 37° C. in DMEM with 10% serum.Supernatants were collected at 48 hours post-transfection. Concentrationof IgG in the supernatant was determined using BioAtla's quantitationELISA. The concentration of the recombinant BAP070-01 was determined byquantitation ELISA to be 3650 ng/ml.

Our first experiment with the BAP070-01 MAb 583 chimera was designed tocompare the MAb 583 chimera to the murine MAb 583 for binding activityto hepcidin-25. Two-fold and then three-fold dilution series of BAP070-1CHO cell supernatant or murine MAb 583 (Lot 10, 0.5 mg/ml) starting at600 ng/ml was incubated in microwell plates with 100 ng of hepcidin-25covalently bound to the maleic anhydride activated wells. Antibodybinding was detected with anti-human IgG (H+L) conjugated with HRP(1:2500) for BAP070-01 supernatant. Antibody binding by the purified MAb583 control was detected using rabbit anti-mouse IgG (H+L) at the samedilution. The reactions were stopped with 1N HCl at 5 minutes after TMBwas added to the wells and read immediately. OD 450 nm value of thereactions was measure with Molecular Device SPECTRAmax Plus (FIG. 29).The OD results shown in the table below and in FIG. 29 demonstrateexcellent binding by the BAP070-01 chimeric MAb 583 and the purified MAb583 control indicating that the BAP070-01 chimera clone was constructedcorrectly and that the murine heavy chain and light chain CDRsfunctioned correctly in the context of the human IgG framework. Thesedata confirm that the cloning, expression of the BAP070-01 in CHO cellculture, and chimera binding activity are sufficiently robust tocontinue the humanization protocol. We conducted additional experimentsto confirm the initial observation.

OD 450 nm values are as follows:

[ng/ml] BAP070-01 Murine MAb 583 600 2.8799 1.5871 300 2.6993 1.285 1001.5046 0.5912 33.3 0.591 0.2297 11 0.2295 0.1035 3.6 0.1257 0.078 1.20.0848 0.0677 0.4 0.0723 0.065

The MAb 583 chimera, BAP070-01, clearly bound to hepcidin-25 to agreater degree that did the serially diluted purified murine MAb 583 inthis experiment, confirming the chimeric antibodies specificity andfunctionality is comparable to the parent murine MAb 583 (FIG. 29).

This initial binding assay was a semi-quantitative assessment of theBAP070-01 chimera since the comparison involves a cell culturesupernatant of a human-mouse chimeric MAb 583 with a purified murine MAb583. Quantitation of chimeric antibody concentration in cell culturesupernatants was performed using a proprietary immunological method andpurified MAb 583 using BCA. The different assay can potentially lead tocomparison of unequal amounts of antibody, and thus signal, in an ELISAcomparison. Despite these caveats, both MAb 583 and the BAP070-01chimera show increasing signal with increasing antibody as is predictedfor this comparison.

583 Chimera Titering by Hepcidin-25 Coated Plate.

To compare the binding activity of BAP070-01 and the empty vector,BAP070 was determined by ELISA. Briefly, human hepcidin-25 (100 ng/well)was covalently bound to maleic anhydride activated microwell platesovernight and the remaining unbound activated sites were blocked as permanufacturer's instructions. Serially diluted culture supernatantscontaining the MAb 583 chimera, BAP070-01, or for the empty vector,BAP070, were added to the microwell plate and incubated at roomtemperature (RT) for 2 hours. Bound chimeric MAb 583 antibody wasdetected using anti-human IgG-HRP with TMB as substrate. The results areshown in FIG. 30.

The data clearly indicates that the BAP070-01 human-mouse chimeric MAbspecifically recognizes human hepcidin-25 with excellent affinity whilethere is no binding to hepcidin-25 by BAP070 CHO supernatant in thisassay (FIG. 30). The data shown in FIGS. 29 and 30 demonstrate a verypositive comparison of purified MAb 583 and the BAP070-01 chimeric MAb583 and the hepcidin-25 specific binding of the of the BAP070-01chimera. The expected ELISA results comparing the supernatants of theBAP070-01 chimera to the negative control BAP070 empty vector wasdemonstrated using hepcidin-25 coated plates to capture functional humanIgG antibodies and anti-human IgG (H+L) antibodies for detection (FIG.30).

Protein G Coated C-ELISA for BAP070-01 583 Chimera

To assess binding of the BAP070-01 chimera to Protein G and the degreeof neutralization of the BAP070-01 MAb 583 by synthetic hepcidin-25 weperformed a C-ELISA assay using NT-biotin hepcidin-25 as the tracer(FIG. 31). This C-ELISA format also was useful to test the binding ofNT-biotin hepcidin-25 tracer to the BAP070-01 chimera and compete withhepcidin-25 for binding to the chimeric MAb 583. To capture BAP070-01chimeric antibodies we coated microwell plates with Protein G (150ng/well) overnight in carbonate coating buffer.

The plate was washed with TBST and CHO cell supernatant containing theBAP070-01 MAb 583 chimera (150 ng/well) was added and allowed to bind tothe Protein G at RT for 1 hour.

The plate was washed with TBST and one (1) ng/well of NT-biotinhepcidin-25 was mixed with different amounts (0-100 ng) of synthetichepcidin-25 standard in TBST, 0.25% BLOTTO and added onto the plate tobind competitively. The plate was washed with TBST and SA-HRP (1:2500)added and allowed to bind for 1 hour.

The plate was washed with TBST and TBS substrate added and the reactionstopped after 10 minutes with stop solution. Absorbance at 450 nm wasmeasured on a spectrophotometer.

Absorbance (OD₄₅₀) for C-ELISA of BAP070-01 is shown in the Table below.

ng hepcidin OD1 OD2 AVG O.D. replicates ratio 100.00 0.0231 0.02330.0232 99 33.33 0.0698 0.0621 0.0660 112 11.11 0.1710 0.1817 0.1764 943.70 0.4657 0.4511 0.4584 103 1.23 0.8793 0.9340 0.9067 94 0.41 1.17511.1974 1.1863 98 0.14 1.2469 1.2306 1.2388 101 0.00001 1.1811 1.23591.2085 96

The results in the table above and in FIG. 31 demonstrate that synthetichepcidin-25 competes for BAP070-10 chimera binding sites competitivelyand that the chimeric MAb 583 antibodies are specific for hepcidin-25and NT-biotin hepcidin-25. The standard curve shown in FIG. 31 wasgenerated using a four parameter logistical regression (Graphpad Prism;San Diego, Calif.). We used Prism to calculate the EC50 for the bindingof hepcidin-25 to BAP070-01 and determined EC50=54 ng/ml (FIG. 31). ThisEC50 is excellent considering that it is derived from a crudesupernatant and not a purified MAb 583 where the EC50 is ≦5.0 ng/ml(FIG. 16).

Neutravidin C-ELISA for BAP070-01 583 Chimera

Another assessment of BAP070-01 chimeric MAb 583 was performed bycoating microwell plates with neutravidin (150 ng/well) overnight incarbonate coating buffer. The plate was washed with TBST. The NT-biotinhepcidin-25 tracer was added at 1 ng/well with different concentrationsof hepcidin-25 (0-100 ng/well) and allowed to bind at RT for 1 hour.Anti-human IgG (H+L)-HRP was used to detect bound BAP070-01 MAb 583chimera in this C-ELISA analysis

The table below shows both duplicate and mean OD₄₅₀ values relative tohepcidin-25 concentrations. The results show that human hepcidin-25clearly neutralizes binding sites on the BAP070-01 chimera and that theNT-biotin hepcidin-25 tracer (hepcidin analog) binds efficiently.

ng hepcidin OD1 OD2 Mean O.D. replicates ratio 100.00 0.2236 0.20290.2133 110 33.33 0.5828 0.6466 0.6147 90 11.11 1.5866 1.4614 1.5240 1093.70 1.7336 1.9523 1.8430 89 1.23 1.6484 1.7580 1.7032 94 0.41 1.84001.7147 1.7774 107 0.14 1.8255 1.5933 1.7094 115 0.00001 1.9864 1.52741.7569 130

FIG. 32 provides graphic data illustrating the results presented in thetable above for competitive binding of the BAP070-01 chimera toNT-biotin hepcidin-25. As expected in any competitive assay, lowersignal is observed with increasing unlabeled antigen (e.g. hepcidin-25)concentration. FIG. 32 shows the two duplicate OD₄₅₀ and the mean OD₄₅₀value for each increasing concentration of hepcidin-25.

Cumulatively, the data we have presented in Example 10 demonstrates thatthe BAP070-01 chimeric Mab 583 retains the high affinity and specificitycharacteristics as the parent murine MAb 583 antibody and that completehumanization of the native murine MAb 583 antibody will yield acandidate therapeutic antibody suitable for pre-clinical and clinicaltesting in humans.

While certain embodiments of the present embodiments have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the embodiments. It should beunderstood that various alternatives to the embodiments of theembodiments described herein may be employed in practicing theembodiments. It is intended that the following claims define the scopeof the embodiments and that methods and structures within the scope ofthese claims and their equivalents be covered thereby.

SEQUENCES

Nucleotide Alignments of CDR-1, CDR-2, and CDR-3 Regions of VariableHeavy and Light Chains of Hepcidin MAbs H32, 583 and 1B1. CDRs areunderlined. Framework regions are not underlined.

Variable Heavy Chain

CDR-1 SEQ ID NO: 1H32 GGTTCTGGCTACACATTCACTGATTATGCTATGCACSEQ ID NO: 2583 GCTTCTGGGTATACCTTCACAAACTATGGAATGAAC SEQ ID NO: 31B1GTCACTGGCTACTCAATCACCAGTGATTATGCCTGGAAC CDR-2 SEQ ID NO: 4H32GGAGTTATTAGTTCTTACTATGGTGATGCTAGCTAC SEQ ID NO: 5583GGCTGGATAAACACCTACACTGGAGAGCCAACATAT SEQ ID NO: 61B1GGCTACATAAGCTACAGTAGTATCACTAACTAC CDR-3 SEQ ID NO: 7H32TACTGTGCAAGATATAGGGGGCTCTGGTACTTCGATGTCTGGGGC SEQ ID NO: 8583TTCTGTACAACGTACGCTACTAGCTGGTACTGGGGC SEQ ID NO: 91B1TACTGTGCTGGTCTTTACTATGTTATGGACCACTGGGGT

Variable Light Chain

CDR-1 H32 SEQ ID NO: 10 TCTAGTAAGAGTCTCCTGCATAGTAATGGCAACACTTACTTGTAT583 SEQ ID NO: 11 GCCAGTGAAAGTGTTGATAGTTATGGCAATAGTTTTATGCAC 1B1SEQ ID NO: 12 GCCAGCTCAAGTGTAAGTTACATGTAC CDR-2 H32 SEQ ID NO: 13GTATATCGGATGTCCAACCTT 583 SEQ ID NO: 14 ATCTATCGTGCATCCAACCTA 1B1SEQ ID NO: 15 ATTTATCTCACATCCAACCTG CDR-3 H32 SEQ ID NO: 16TATTGTATGCAACATCTAGAATATCCTTTCACGTTCGGT 583 SEQ ID NO: 17TACTGTCAGCAAAGTAATGAGGATCTGACGTTCGGT 1B1 SEQ ID NO: 18TACTGCCAGCAGTGGAGTAGTGACCCTTTCACGTTCGGC

Human hepcidin peptide (hepcidin-25, hep-25, Hep-25, hHepcidin-25):

(25 aa)  SEQ ID NO: 19 DTHFPICIFCCGCCHRSKCGMCCKT

Mouse hepcidin-1 peptide (mhepcidin-1, mhep-1, mHep-1, mHepcidin-1):

(25 aa) SEQ ID NO: 20: DTNFPICIFCCKCCNNSQCGICCKT

Rat hepcidin peptide (rhepcidin, rhep, rHep, rHepcidin):

SEQ ID NO: 21: (25aa) DTNFPICLFCCKCCKNSSCGLCCIT

Human hepcidin-20 peptide (hepcidin-20, hep-20, Hep-25, hHepcidin-20):

SEQ ID NO: 22: (20aa) ICIFCCGCCHRSKCGMCCKT

Human hepcidin 22 peptide (hepcidin-22, hep-22, Hep-22, hHepcidin-22):

SEQ ID NO: 23: (23aa) HFPICIFCCGCCHRSKCGMCCKT

Human hepcidin-9 peptide (hepcidin-9, hep-9, Hep-9, hHepcidin-9):

SEQ ID NO: 24 (9aa) DTHFPICIF

Human hepcidin-5 peptide (hepcidin-5, hep-5, Hep-5, hHepcidin-5):

SEQ ID NO: 25 (5aa) DTHFP

Human hepcidin 10-25 peptide (hepcidin 10-25, hep 10-25, Hep 10-25,hHepcidin 10-25):

SEQ ID NO: 26 (16aa) CCGCCHRSKCGMCCKT

DNP-human hepcidin-9 KLH peptide (DNP-hepcidin-9-KLH, DNP-hep-9-KLH,DNP-Hep-9-KLH, DNP-hHepcidin-9-KLH):

SEQ ID NO: 27 DNP-DTHFPIC(KLH-SMCC)-IF

IgG1 heavy chain variable region [Homo sapiens] GenBank: AAK62671.1

(SEQ ID NO: 29) LLESGPGLLKPSETLSLTCTVSGGSMINYYWSWIRQPPGERPQWLGHIIYGGTTKYNPSLESRITISRDISKSQFSLRLNSVTAADTAIYYCARVAIGVSGFLNYYYYMDVWGSGTAVTVSS

IgG1 heavy chain variable region [Homo sapiens] GenBank: AAK19936.1

(SEQ ID NO: 30) QVQLQQWGAGLLKPSETLSRTCAVYGGSFSDDYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVDTSEKQFSLKLSSVTAADTAVYYCARRNDWYPFDYWDEGILVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSW

IgG1 [Mus musculus] GenBank: BAA23565.1

(SEQ ID NO: 31) QVQLQQSGAELMKPGASVNISCKASGYIFSSYWIEWVKQRPGHGLEWIGEILPGSGNIKYNEKFKGKAIFTVETSSNTAYMQLSSLTSEDSAVYFCAKTD YYASGYGFDYWGQGTTVTVSS

Immunoglobulin kappa light chain variable region [Mus musculus] GenBank:ABE03823.1

(SEQ ID NO: 32) DIVMTQSPASLDVSLGQRATISCRASKSVSTSGYSYMNWYQQKPGQPPKLLIYLASSLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSREPPP TFGGGTKLEIKRAD

Immunoglobulin kappa light chain variable region [Mus musculus] GenBank:ABE03821.1

(SEQ ID NO: 33) DVVMTQSPLTLSVTIGQPASISCKSSQSLLANNGRTYLNWLLQRPGQSPKRLIYLVSTLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFP LTFGAGTKLELKRAD

Immunoglobulin IgG1 light chain variable region [Homo sapiens] GenBank:AAK62672.1

(SEQ ID NO: 34) LTQSPATLSLSPGERATLSCRASQSVGRNLGWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSDWPRTFGQGTK VEIKR Mouse VK(SEQ ID NO: 35) QIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWSSNPLTFGAG TKLELK Mouse VH(SEQ ID NO: 36) EVKLEESGGGLVQPGGSMKLSCAASGFTFSDAWMDWVRQSPEKGLEWVAEIRSKASNHATYYAESVKGRFTISRDDSKSSVYLQMNSLRAEDTGIYYCTR WRRFFDSWGQGTTLTVSS

Variable Heavy and Light Chain Nucleic Acid and Amino Acid Sequences ofHepcidin MAbs H32, 583 and 1B1.

H32 VH (SEQ ID NO: 41)GGTTCTGGCTACACATTCACTGATTATGCTATGCACGGAGTTATTAGTTCTTACTATGGTGATGCTAGCTACTACTGTGCAAGATATAGGGGGCTCTGGT ACTTCGATGTCTGGGGCH32 VH (SEQ ID NO: 42) Gly Ser Gly Tyr Thr Phe Thr Asp Tyr Ala Met HisGly Val Ile Ser Ser Tyr Tyr Gly Asp Ala Ser TyrTyr Cys Ala Arg Tyr Arg Gly Leu Trp Tyr Phe Asp Val Trp Gly 583 VH(SEQ ID NO: 43) GCTTCTGGGTATACCTTCACAAACTATGGAATGAACGGCTGGATAAACACCTACACTGGAGAGCCAACATATTTCTGTACAACGTACGCTACTAGCTGGT ACTGGGGC 583 VH(SEQ ID NO: 44) Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly Met AsnGly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr TyrPhe Cys Thr Thr Tyr Ala Thr Ser Trp Tyr Trp Gly 1B1 VH (SEQ ID NO: 45)GTCACTGGCTACTCAATCACCAGTGATTATGCCTGGAACGGCTACATAAGCTACAGTAGTATCACTAACTACTACTGTGCTGGTCTTTACTATGTTATGG ACCACTGGGGT 1B1 VH(SEQ ID NO: 46) Val Thr Gly Tyr Ser Ile Thr Ser Asp Tyr Ala TrpAsn Gly Tyr Ile Ser Tyr Ser Ser Ile Thr Asn TyrTyr Cys Ala Gly Leu Tyr Tyr Val Met Asp His Trp Gly H32 VL(SEQ ID NO: 47) TCTAGTAAGAGTCTCCTGCATAGTAATGGCAACACTTACTTGTATGTATATCGGATGTCCAACCTTTATTGTATGCAACATCTAGAATATCCTTTCACGT TCGGT H32 VL(SEQ ID NO: 48) Ser Ser Lys Ser Leu Leu His Ser Asn Gly Asn ThrTyr Leu Tyr Val Tyr Arg Met Ser Asn Leu Tyr CysMet Gln His Leu Glu Tyr Pro Phe Thr Phe Gly 583 VL (SEQ ID NO: 49)GCCAGTGAAAGTGTTGATAGTTATGGCAATAGTTTTATGCACATCTATCGTGCATCCAACCTATACTGTCAGCAAAGTAATGAGGATCTGACGTTCGGT 583 VL (SEQ ID NO: 50)Ala Ser Glu Ser Val Asp Ser Tyr Gly Asn Ser PheMet His Ile Tyr Arg Ala Ser Asn Leu Tyr Cys GlnGln Ser Asn Glu Asp Leu Thr Phe Gly 1B1 VL (SEQ ID NO: 51)GCCAGCTCAAGTGTAAGTTACATGTACATTTATCTCACATCCAACCTGTACTGCCAGCAGTGGAGTAGTGACCCTTTCACGTTCGGC 1B1 VL (SEQ ID NO: 52)Ala Ser Ser Ser Val Ser Tyr Met Tyr Ile Tyr LeuThr Ser Asn Leu Tyr Cys Gln Gln Trp Ser Ser Asp Pro Phe Thr Phe Gly

CDR-1, CDR-2, and CDR-3 polynucleotide sequences of Variable Heavy andLight Chains of Hepcidin MAbs H32, 583 and 1B1.

Variable Heavy Chain CDR-1 SEQ ID NO: 55 H32 GGCTACACATTCACTGATTATGCTSEQ ID NO: 56 583 GGGTATACCTTCACAAACTATGGA SEQ ID NO: 57 1B1GGCTACTCAATCACCAGTGATTATGCC CDR-2 SEQ ID NO: 58 H32ATTAGTTCTTACTATGGTGATGCT SEQ ID NO: 59 583 ATAAACACCTACACTGGAGAGCCASEQ ID NO: 60 1B1 ATAAGCTACAGTAGTATCACT CDR-3 SEQ ID NO: 61 H32GCAAGATATAGGGGGCTCTGGTACTTCGATGTC SEQ ID NO: 62 583ACAACGTACGCTACTAGCTGGTAC SEQ ID NO: 63 1B1 GCTGGTCTTTACTATGTTATGGACCACVariable Light Chain CDR-1 SEQ ID NO: 64 H32AAGAGTCTCCTGCATAGTAATGGCAACACTTAC SEQ ID NO: 65 583GAAAGTGTTGATAGTTATGGCAATAGTTTT SEQ ID NO: 66 1B1 TCAAGTGTAAGTTAC CDR-2SEQ ID NO: 67 H32 CGGATGTCC SEQ ID NO: 68 583 CGTGCATCC SEQ ID NO: 691B1 CTCACATCC CDR-3 SEQ ID NO: 70 H32 ATGCAACATCTAGAATATCCTTTCACGSEQ ID NO: 71 583 CAGCAAAGTAATGAGGATCTGACG SEQ ID NO: 72 1B1CAGCAGTGGAGTAGTGACCCTTTCACG

REFERENCES

-   Hunter et al., J. Biol. Chem., 277:37597-37603 (2002)-   Kemna et al., Blood, 106:1864-1866, 2005-   Kilpatrick K E, Wring S A, Walker D H, et al. 1997. Rapid    Development of Monoclonal Antibodies Using Repetitive Immunization,    Multiple Sites.-   Krause et al., FEBS Lett. 480:147 (2000)-   Lauth et al., J. Biol. Chem., 280:9272-9282 (2005)-   Nemeth et al., J. Clin. Invest., 113:1271-1276, 2004-   Nemeth et al., Blood, 101:2461-2463, 2003-   Nicolas et al., Nat. Genet., 34:97-101, 2003-   Nicolas et al., Proc. Natl. Acad. Sci. USA, 99:4596-4601, 2002-   Nicolas et al., Proc. Natl. Acad. Sci. USA, 98:8780-8785, 2001.-   Nicolas et al., J. Clin. Invest., 110:1037-1044, 2002-   Park et al., J. Biol. Chem. 276:7806 (2001)-   Pigeon et al., J. Biol. Chem. 276:7811 (2001)-   Rivera et al., Blood, 105:1797-1802, 2005-   Roetto et al., Nat. Genet., 33:21-22, 2003-   Weinstein et al., Blood, 100:3776-36781, 2002 The N-terminus of    hepcidin is essential for its interaction with ferroportin:-   Nemeth et al., Blood, 107(1):328-333, 2006.

What is claimed is:
 1. An antibody, or antigen-binding fragment thereof,that specifically binds to hepcidin (Hep) or a hepcidin peptide, whereinthe antibody, or antigen-binding fragment thereof, comprises a heavychain CDR1 encoded by SEQ ID NO: 57, a heavy CDR2 encoded by SEQ ID NO:60, a heavy chain CDR3 encoded by SEQ ID NO: 63, a light chain CDR1encoded by SEQ ID NO: 66, a light CDR2 encoded by SEQ ID NO: 69, and alight chain CDR3 encoded by SEQ ID NO:
 72. 2. An antibody, orantigen-binding fragment thereof, that specifically binds to hepcidin ora hepcidin peptide, that comprises a heavy chain variable region havingan amino acid sequence of SEQ ID NO: 46 and a light chain variableregion having an amino acid sequence of SEQ ID NO:
 52. 3. The antibody,or antigen-binding fragment thereof, of claim 2, wherein the antibody,or antigen-binding fragment thereof, comprises a heavy chain CDR1encoded by SEQ ID NO: 57, a heavy CDR2 encoded by SEQ ID NO: 60, a heavychain CDR3 encoded by SEQ ID NO: 63, a light chain CDR1 encoded by SEQID NO: 66, a light CDR2 encoded by SEQ ID NO: 69, and a light chain CDR3encoded by SEQ ID NO:
 72. 4. The antigen-binding fragment of claim 2,wherein the antigen-binding fragment is a Fab fragment, a Fab′ fragment,a F(ab′)₂ fragment, an Fv fragment, an scFv fragment or a single chainbinding polypeptide.
 5. The antibody, or antigen-binding fragmentthereof, of claim 2, comprising an IgG1 or an IgG4 heavy chain variableregion; and an IgG1 or an IgG4 light chain variable region.
 6. Theantibody, or antigen-binding fragment thereof, of claim 2, thatspecifically binds to an epitope comprising amino acid residues 1-9 ofhepcidin.
 7. The antibody of claim 2, wherein the antibody is amonoclonal antibody, a chimeric antibody, a human antibody, a bivalentantibody, a multispecific antibody, a maxibody, a nanobody or ahumanized antibody.
 8. A pharmaceutical composition comprising anantibody, or antigen-binding fragment, of claim 2, and an acceptablecarrier or excipient.
 9. A method of treating a disorder of ironhomeostasis associated with elevated hepcidin levels in a subject inneed thereof, comprising administering to the subject a pharmaceuticalcomposition of claim
 8. 10. A method of reducing hepcidin activityassociated with elevated hepcidin levels in a subject in need thereof,comprising administering to the subject a pharmaceutical composition ofclaim
 8. 11. A method of treating hemochromatosis associated withelevated hepcidin levels in a subject in need thereof, comprisingadministering to the subject a pharmaceutical composition of claim 8.12. A method of treating a subject with an elevated level of hepcidin,comprising administering to the subject a pharmaceutical composition ofclaim
 8. 13. The method of claim 12, further comprising administering tothe subject a erythropoiesis stimulator, wherein the erythropoiesisstimulator is selected from the group consisting of erythropoietin, anerythropoietin variant and an antibody that binds erythropoietin. 14.The method of claim 13, wherein the antibody, or antigen-bindingfragment thereof, that specifically binds to hepcidin, or a hepcidinpeptide, and the erythropoiesis stimulator are administered concurrentlyor sequentially.
 15. The method of claim 12, wherein administeringcomprises an injection.
 16. The method of claim 15, wherein injection isan intravenous injection, a subcutaneous injection, an intramuscularinjection, or a spinal injection into a cerebrospinal fluid.
 17. Amethod of treating a subject with iron refractory iron deficiency anemia(IRIDA), comprising administering to the subject a pharmaceuticalcomposition of claim
 8. 18. A method of treating anemia of inflammationin a subject in need thereof, comprising administering to the subject apharmaceutical composition of claim
 8. 19. A method of treating aninflammatory disease associated with elevated hepcidin levels in asubject in need thereof, comprising administering to the subject apharmaceutical composition of claim
 8. 20. A method of treating aninfection associated with elevated hepcidin levels in a subject in needthereof, comprising administering to the subject a pharmaceuticalcomposition of claim
 8. 21. The method of claim 20, wherein saidinfection is a bacterial infection, fungal infection, or a viralinfection.
 22. A method of treating anemia associated with elevatedhepcidin levels in a subject in need thereof, comprising administeringto the subject a pharmaceutical composition of claim
 8. 23. A containerthat comprises an antibody, or antigen-binding fragment thereof, ofclaim
 2. 24. The container of claim 23, wherein the container is a vial,a syringe, a bottle or an ampoule.
 25. A kit for treating a disorderassociated with elevated hepcidin levels or a disorder of ironhomeostasis associated with elevated hepcidin levels, that comprises anantibody, or an antigen-binding fragment thereof, of claim 2, and anerythropoiesis stimulator.
 26. A kit for treating a disorder associatedwith elevated hepcidin levels or a disorder of iron homeostasisassociated with elevated hepcidin levels, that comprises an antibody, oran antigen-binding fragment thereof, of claim 2, and an erythropoiesisstimulator, wherein a level of hepcidin in a subject having the disorderis in a physiologically normal range, but elevated relative to a plasmairon levels or to % TSAT (transferring saturation).
 27. A kit fortreating a disorder associated with elevated hepcidin levels or adisorder of iron homeostasis associated with elevated hepcidin levels,that comprises an antibody, or an antigen-binding fragment thereof, ofclaim 2, and a label attached to, or packaged within, the container, thelabel describing use of the antibody, the antigen-binding fragmentthereof, with an erythropoiesis stimulator.
 28. A kit for treating adisorder associated with elevated hepcidin levels, that comprises anantibody, or an antigen-binding fragment thereof, of claim 2, anerythropoiesis stimulator and a label attached to or packaged with thecontainer, the label describing use of the erythropoiesis stimulatorwith the antibody, or the antigen-binding fragment thereof.